CD80 variant immunomodulatory proteins and uses thereof

ABSTRACT

Provided herein are variant CD80 polypeptides, immunomodulatory proteins comprising variant CD80 polypeptides, and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of immunological and oncological conditions. Compositions and methods for making and using such proteins are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/088,802, filed Sep. 26, 2018, which is a U.S. National StageApplication of International Application No. PCT/US2017/027817, filedApr. 14, 2017, which claims priority from U.S. provisional applicationNo. 62/323,595 filed Apr. 15, 2016, entitled “CD80 VariantImmunomodulatory Proteins and Uses Thereof,” U.S. provisionalapplication No. 62/394,743 filed Sep. 14, 2016, entitled “CD80 VariantImmunomodulatory Proteins and Uses Thereof,” U.S. provisionalapplication No. 62/410,844 filed Oct. 20, 2016, entitled “CD80 VariantImmunomodulatory Proteins and Uses Thereof,” U.S. provisionalapplication No. 62/472,570 filed Mar. 16, 2017, entitled “CD80 VariantImmunomodulatory Proteins and Uses Thereof,” and U.S. provisionalapplication No. 62/475,201 filed Mar. 22, 2017, entitled “CD80 VariantImmunomodulatory Proteins and Uses Thereof,” the contents of each ofwhich are incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled761612000410SubSeqList.txt, created Jul. 21, 2021 which is 816,024 bytesin size. The information in the electronic format of the SequenceListing is incorporated by reference in its entirety.

FIELD

The present disclosure relates to therapeutic compositions formodulating immune response in the treatment of cancer and immunologicaldiseases. In some aspects, the present disclosure relates to particularvariants of CD80 that exhibit altered affinity for a cognate bindingpartner.

BACKGROUND

Modulation of the immune response by intervening in the processes thatoccur in the immunological synapse (IS) formed by and betweenantigen-presenting cells (APCs) or target cells and lymphocytes is ofincreasing medical interest. Mechanistically, cell surface proteins inthe IS can involve the coordinated and often simultaneous interaction ofmultiple protein targets with a single protein to which they bind. ISinteractions occur in close association with the junction of two cells,and a single protein in this structure can interact with both a proteinon the same cell (cis) as well as a protein on the associated cell(trans), likely at the same time. Although therapeutics are known thatcan modulate the IS, improved therapeutics are needed. Provided areimmunomodulatory proteins, including soluble proteins or transmembraneimmunomodulatory proteins capable of being expressed on cells, that meetsuch needs.

SUMMARY

In some embodiments, provided herein is a variant CD80 polypeptidecontaining an IgV domain or a specific binding fragment thereof, an IgCdomain or a specific binding fragment thereof, or both, wherein thevariant CD80 polypeptide containing one or more amino acid modificationsin an unmodified CD80 or specific binding fragment thereof,corresponding to position(s) 4, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,20, 21, 22, 24, 25, 27, 28, 29, 30, 31, 33, 36, 37, 38, 40, 41, 42, 43,44, 47, 48, 50, 52, 53, 54, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 72, 74, 76, 77, 80, 81, 83, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 99, 102, 103, 104, 108, 107, 109, 110, 114, 115, 116, 117,118, 120, 121, 122, 126, 127, 128, 129, 130, 133, 137, 140, 142, 143,144, 148, 149, 152, 154, 160, 162, 164, 168, 169, 174, 175, 177, 178,183, 178, 185, 188, 190, 192, 193, or 199 with reference to numbering ofSEQ ID NO: 28. In some embodiments, the one or more amino acidmodifications includes one or more amino acid substitution, insertion ordeletion. In some embodiments, the unmodified CD80 is a mammalian CD80or a specific binding fragment thereof. In some embodiments, theunmodified CD80 is a human CD80 or a specific binding fragment thereof.In some embodiments, the variant CD80 polypeptide includes: the IgVdomain or a specific binding fragment thereof; and the IgC domain or aspecific binding fragment thereof. In some embodiments, the unmodifiedCD80 includes (i) the sequence of amino acids set forth in SEQ ID NO:28,(ii) a sequence of amino acids that has at least 95% sequence identityto SEQ ID NO:28; or (iii) is a portion thereof comprising an IgV domainor IgC domain or specific binding fragments thereof.

In some embodiments of any one of the variant CD80 polypeptides, thespecific binding fragment of the IgV domain or the IgC domain has alength of at least 50, 60, 70, 80, 90, 100, 110 or more amino acids; thespecific binding fragment of the IgV domain includes a length that is atleast 80% of the length of the IgV domain set forth as amino acids35-135 of SEQ ID NO:1; or the specific binding fragment of the IgCdomain includes a length that is at least 80% of the length of the IgCdomain set forth as amino acids 145-230 of SEQ ID NO:1. In someembodiments, the variant CD80 polypeptide contains up to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acidmodifications, optionally amino acid substitutions. In some embodiments,the variant CD80 polypeptide includes a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 28, or a specificbinding fragment thereof. In some embodiments, the variant CD80 exhibitsaltered binding specificity to the ectodomain of CD28, PD-L1, or CTLA-4compared to the unmodified CD80. In some instances, the altered bindingis altfered binding affinity and/or altered binding selectivity.

In some embodiments of any one of the variant CD80 polypeptides, the oneor more amino acid substitution is V4M, K9E, E10R, V11S, A12G, A12T,A12V, T13N, L14A, S15V, S15F, C16S, C16G, C16L, G17W, H18L, H18R, H18Y,V20L, S21P, V22A, E24G, L25P, Q27R, T28A, T28S, R29C, R29D, R29H, R29V,I30V, Y31F, Y31H, Y31L, Q33H, K36E, K36G, K37E, K37Q, M38I, M38L, M38T,M38V, L40M, T41A, T41G, T41D, T41I, M42T, M43I, M43Q, M43R, M43V, S44P,M47T, N48I, N48D, W50G, E52G, Y53C, K54M, F59L, F59S, D60V, I61N, T62S,N63S, N64S, L65H, S66H, I67F, I67T, V68A, V68M, I69T, L70Q, L70P, L70R,L72P, P74L, D76G, E77G, E77K, Y80N, E81A, E81R, E81V, V83A, V83I, L85I,L85R, K86E, Y87N, E88D, E88G, K89E, K89N, K89R, D90K, D90L, D90N, A91E,A91G, A91S, A91T, F92L, F92N, F92P, F92Y, K93I, K93E, K93Q, K93R, K93V,R94G, R94L, R94F, E95K, H96R, L97R, E99G, E99D, L102S, S103L, S103P,V104A, V104L, D107N, F108L, P109S, P109H, T110A, S114T, D115G, F116S,F116L, E117V, E117G, I118V, I118A, I118T, T120S, S121P, N122S, I126L,I126V, I127T, C128Y, C128R, S129L, S129P, T130A, G133D, P137L, S140T,L142S, E143G, N144S, N144D, L148S, N149D, N149S, N152T, T154I, T154A,E160G, E162G, Y164H, S168G, K169E, K169I, K169S, M174T, M174V, T175A,N177S, H178R, L183H, K185E, H188D, H188Q, R190S, N192D, Q193L, T199S, ora conservative amino acid substitution thereof.

In some embodiments, the variant CD80 polypeptide contains an IgV domainor an IgC domain, or a specific binding fragment thereof, comprising oneor more amino acid deletions in an unmodified CD80 or specific bindingfragment thereof. In some embodiments, the deletion corresponds toposition 43 of SEQ ID NO: 28.

In some embodiments, the one or more amino acid substitution isV4M/L70Q/A91G/T120S/T130A, A12T/H18L/M43V/F59L/E77K/P109S/I118T,A12V/S15F/Y31H/T41G/T130A/P137L/N152T, V20L/L70Q/A91S/T120S/T130A,V22A/L70Q/S121P, E24G/L25P/L70Q/T120S, T28S/L70Q/A91G/E95K/T120S/T130A,E24G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/H96R,R29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S,R29H/E52G/L70R/E88G/A91G/T130A, R29H/E52G/T120S/T130A,R29V/Y31F/K36G/M38L/M43Q/E81R/V83I/L85I/K89R/D90L/A91E/F92N/K93Q/R94G,R29V/M43Q/E81R/L85I/K89R/D90L/A91E/F92N/K93Q/R94G,Y31H/T41G/L70Q/A91G/T120S/T130A, K36G, K36G/K37Q/M38I/L40M,K36G/K37Q/M38I/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/E99G/T130A/N149S,K36E/I67T/L70Q/A91G/T120S/T130A/N152T, K37E/F59S/L70Q/A91G/T120S/T130A,M38T/L70Q/E77G/A91G/T120S/T130A/N152T,M38V/T41D/M43I/W50G/D76GN83A/K89E/T120S/T130A, T41I/A91G,S44P/L70Q/A91G/T130A, E52G/L70Q/A91G/T120S/T130A, K54M/A91G/T120S,D60V/A91G/T120S/T130A, N63S/L70Q/A91G/T120S/T130A,S66H/D90G/T110A/F116L, I67F/L70R/E88G/A91G/T120S/T130A,I67T/L70Q/A91G/T120S, V68A/T110A, V68M/L70P/L72P/K86E,L70Q/A91G/T110A/T120S/T130A, L70Q/E81A/A91G/T120S/I127T/T130A,L70Q/Y87N/A91G/T130A, L70Q/A91G, L70Q/A91G/E117G/T120S/T130A,L70Q/A91G/T120S/T130A, L70Q/A91G/T130A, L70Q/A91G/I118A/T20S/T130A,L70R/A91G/T120S/T130A, E88D/K89R/D90K/A91G/F92Y/K93R, K89E/T130A,K89R/D90K/A91G/F92Y/K93R, E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S,K89R/D90K/A91G/F92Y/K93R/N122S/N177S, A91G, A91G/F92L/F108L/T120S,A91G/L102S, A91G/S103P, A91G/T120S/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/M174T,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/H188D,H18R/R29D/Y31L/Q33H/K36G/K37E/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/E143G/K169E/M174V/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/F108L/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/C128Y/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/T130A/K169E,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93I/R94L/L97R/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93I/R94L/L97R/T130A/L148S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/I61N/E81V/L85R/K89N/A91T/F92P/K93V/R94F/V104A/T120S/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/F92P/K93V/R94F/I118V/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/T175A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/F116S/T130A/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/L142S/H188D,C16S/H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T110A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/A91G/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/D76G/A91G/S103L/T120S/I127T/T130A, Q33deleted/Y53C/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/S129L/H188D,K9E/E10R/V11S/A12G/T13N/K14A/S15V/C16L/G17W/H18Y/Y53C/L70Q/D90G/T130A/N149D/N152T/H188D,H18L/R29D/Y31L/Q33H/K36G/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,K89E/K93E/T130A,S21P/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N48I/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/P109H/I126L/K169I,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/P74L/Y80N/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R,S21P/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/P74L/Y80N/E81V/L85R/K89N/D90N/A91T/F92P/K93V/R94L/T130A/N149S/E162G,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/R190S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/P74L/Y80N/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/R190S,C16GN22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/D76G/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169I/H178R/N192D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/E117V/I118T/N149S/S168G/H188Q,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N64S/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118T/T130A/N149S/K169I,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/D115G/I118T/T130A/G133D/N149S,S129P, A91G/S129P, I69T/L70Q/A91G/T120S, Y31H/S129P,T28A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/V104L/T130A/N149S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/N149S/H188Q,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/N149S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/T154I,A12G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/T130A/L183H,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I/Q193L,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/T130A/N149S/K169I,I118T/C128R, Q27R/R29C/M42T/S129P/E160G, S129P/T154A,S21P/L70Q/D90G/T120S/T130A, L70Q/A91G/N144D,L70Q/A91G/I118A/T120S/T130A/K169E,V4M/L70Q/A91G/I118V/T120S/T130A/K169E,L70Q/A91G/I118V/T120S/T130A/K169E, L70Q/A91G/I118V/T120S/T130A,V20L/L70Q/A91S/I118V/T120S/T130A, L70Q/A91G/E117G/I118V/T120S/T130A,A91G/I118V/T120S/T130A, L70R/A91G/I118V/T120S/T130A/T199S,L70Q/E81A/A91G/I118V/T120S/I127T/T130A,T28S/L70Q/A91G/E95K/I118V/T120S/I126V/T130A/K169E,N63S/L70Q/A91G/S114T/I118V/T120S/T130A,K36E/I67T/L70Q/A91G/I118V/T120S/T130A/N152T,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E,K37E/F59S/L70Q/A91G/I118V/T120S/T130A/K185E,D60V/A91G/I118V/T120S/T130AK169E, K54M/L70Q/A91G/Y164H/T120S,M38T/L70Q/E77G/A91G/I118V/T120S/T130A/N152T,Y31H/T41G/M43L/L70Q/A91G/I118V/T120S/I126V/T130A, L65H/D90G/T110A/F116L,R29H/E52G/D90N/I118V/T120S/T130A, I67T/L70Q/A91G/I118V/T120S,L70Q/A91G/T110A/I118V/T120S/T130A,M38V/T41D/M43I/W50G/D76GN83A/K89E/I118V/T120S/I126V/T130A,A12V/S15F/Y31H/M38L/T41G/M43L/D90N/T130A/P137L/N149D/N152T,I67F/L70R/E88G/A91G/I118V/T120S/T130A,E24G/L25P/L70Q/A91G/I118V/T120S/N152T, A91G/F92L/F108L/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S,K36G/K37Q/M38I/L40M/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/E99G/T130A/N149S,K36G/L40M,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/M174T,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N48D/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,H18R/R29D/Y31L/Q33H/K36G/K37E/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/E143G/K169E/M174V/H188D,R29D/I30V/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/F108L/I118V/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/N149D/K169E/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/C128Y/T130A/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/E99D/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/I61N/E81V/L85R/K89N/A91T/F92P/K93V/R94F/V104A/I118V/T120S/I126V/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118V/T120S/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/L142S/H188D,C16S/H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T110A/I118V/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/A91G/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/D76G/A91G/S103L/I118V/T120S/I127T/T130A,Y53C/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E,Y53C/L70Q/D90G/T130A/N149D/N152T/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,orH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide contains the IgV domain or a specific fragmentthereof and/or the IgC domain or a specific fragment thereof. In someembodiments of any one of the variant CD80 polypeptides, the variantCD80 polypeptide contains the IgV domain or a specific binding fragmentthereof. In some embodiments, the IgV domain or specific bindingfragment thereof is the only CD80 portion of the variant CD80polypeptide. In some embodiments, the IgC domain or specific bindingfragment thereof is the only CD80 portion of the variant CD80polypeptide.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide includes the sequence of amino acids set forthin any of SEQ ID NOS: 55-108, 280-346, 414-475 or a specific bindingfragment thereof, or a sequence of amino acids that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% sequence identity to any of SEQ ID NOS: 55-108, 280-346, 414-475 ora specific binding fragment thereof and that contains the one or more ofthe amino acid substitutions. In some embodiments, the variant CD80polypeptide includes the IgV domain or a specific binding fragmentthereof. In some embodiments, the IgV domain or specific fragmentthereof is the only CD80 portion of the variant CD80 polypeptide. Insome embodiments, the IgC domain or specific fragment thereof is theonly CD80 portion of the variant CD80 polypeptide. In some embodimentsof any one of the variant CD80 polypeptides, the variant CD80polypeptide includes the sequence of amino acids set forth in any of SEQID NOS: 153-195, 347, 373-386, 476-477 or a specific binding fragmentthereof, a sequence of amino acids that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequenceidentity to any of SEQ ID NOS: 153-195, 347, 373-386, 476-477 or aspecific binding fragment thereof and that contains the one or more ofthe amino acid substitutions.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of CD28,PD-L1, or CTLA-4 with increased affinity compared to the unmodified CD80polypeptide. In some of any such embodiments, the variant polypeptidespecifically binds to the ectodomain of CD28, PD-L1 or CTLA-4 withincreased selectivity compared to the binding of the unmodified CD80 forthe ectodomain. In some aspects, the increased selectivity includes agreater ratio for one cognate binding partner selected from among CD28,PD-L1 and CTLA-4 versus another of the cognate binding partner comparedto the ratio of binding of the unmodified CD80 polypeptide for the onecognate binding partner versus the another of the cognate bindingpartner.

In some embodiments, the variant polypeptide specifically binds to theectodomain of CD28 with increased selectivity compared to the binding ofthe unmodified CD80 for the ectodomain of CD28. In some aspects, theincreased selectivity includes a greater ratio for binding CD28 versusPD-L1 or CTLA-4 compared to the ratio of binding of the unmodified CD80polypeptide for CD28 versus PD-L1 or CTLA-4. In some cases, the ratio isgreater by at least or at least about 1.5-fold, 2.0-fold, 3.0-fold,4.0-fold. 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-foldor more.

In some embodiments, the variant CD80 polypeptide specifically binds tothe ectodomain of CD28 with increased affinity compared to theunmodified CD80 polypeptide. In some embodiments, the increased affinityto the ectodomain is increased more than 1.2-fold, 1.5-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold or 60-fold compared to the bindingaffinity of the unmodified CD80 for the ectodomain.

In some embodiments of any one of the variant CD80 polypeptides, the oneor more amino acid substitutions corresponds to position(s) 12, 18, 20,29, 31, 36, 40, 41, 43, 52, 59, 60, 63, 67, 70, 77, 81, 87, 88, 89, 90,91, 92, 93, 107, 109, 114, 117, 118, 120, 122, 127, 130, 144, 169, 177or 199 with reference to numbering of SEQ ID NO: 28. In someembodiments, the one or more amino acid substitution is selected fromthe group consisting of A12T, H18L, V20L, R29H, Y31H, K36G, L40M, T41G,T41I, M43V, E52G, F59L, D60V, N63S, I67T, L70Q, L70R, E77K, E81A, Y87N,E88D, E88G, K89E, K89R, D90K, D90N, A91G, A91S, F92Y, K93R, D107N,P109S, S114T, E117G, I118A, I118T, I118V, T120S, I127T, T130A, N144D,K169E, N177S and T199S and conservative amino acid substitutionsthereof. In some embodiments, the one or more amino acid substitution isA12T/H18L/M43V/F59L/E77K/P109S/I118T, V20L/L70Q/A91S/T120S/T130A,V20L/L70Q/A91S/I118V/T120S/T130A,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,T41I/A91G, E52G/L70/A91G/T120S/T130A,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E, D60V/A91G/T120S/T130A,D60V/A91G/I118V/T120S/T130A/K169E, N63S/L70Q/A91G/T120S/T130A,N63S/L70Q/A91G/S114T/I118V/T120S/T130A, I67T/L70Q/A91G/T120S,I67T/L70Q/A91G/I118V/T120S, L70Q/E81A/A91G/T120S/I127T/T130A,L70Q/E81A/A91G/I118V/T120S/I127T/T130A, L70Q/Y87N/A91G/T130A, L70Q/A91G,L70Q/A91GN144D, L70Q/A91G/E117G/T120S/T130A,L70Q/A91G/E117G/I118V/T120S/T130A, L70Q/A91G/I118A/T120S/T130A,L70Q/A91G/I118A/T120S/T130A/K169E, L70Q/A91G/T120S/T130A,L70Q/A91G/I118V/T120S/T130A/K169E, L70R/A91G/T120S/T130A,L70R/A91G/I118V/T120S/T130A/T199S, E88D/K89R/D90K/A91G/F92Y/K93R,K89R/D90K/A91G/F92Y/K93R, or K89R/D90K/A91G/F92Y/K93R/N122S/N177S.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of PD-L1with increased affinity compared to the unmodified CD80 polypeptide. Insome cases, the increased affinity to the ectodomain is increased morethan 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-foldcompared to the binding affinity of the unmodified CD80 for theectodomain. In some of any such embodiments, the variant polypeptidespecifically binds to the ectodomain of PD-L1 with increased selectivitycompared to the binding of the unmodified CD80 for the ectodomain. Insome cases, the increased selectivity includes a greater ratio forbinding PD-L1 versus CD28 or CTLA-4 compared to the ratio of binding ofthe unmodified CD80 polypeptide for PD-L1 versus CD28 or CTLA-4. In someinstances, the ratio is greater by at least or at least about 1.5-fold,2.0-fold, 3.0-fold, 4.0-fold. 5-fold, 10-fold, 15-fold, 20-fold,30-fold, 40-fold, 50-fold or more.

In some embodiments, the one or more amino acid substitutionscorresponds to position(s) 12, 18, 29, 31, 33, 36, 38, 40, 41, 42, 43,47, 59, 67, 70, 77, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 109, 118,120, 122, 144, 148, 149, or 177 with reference to numbering of SEQ IDNO: 28. In some embodiments, the one or more amino acid substitution isselected from the group consisting of A12T, H18L, R29D, R29H, Y31H,Y31L, Q33H, K36G, M38I, L40M, T41A, T41G, M42T, M43R, M43V, M47T, F59L,I67T, L70Q, E77K, E81V, L85R, Y87N, E88D, E88G, K89E, K89N, K89R, D90K,D90N, A91G, A91T, F92P, F92Y, K93R, K93V, R94L, P109S, I118T, I118V,T120S, N122S, N144S, L148S, N149S, and N177S, and conservative aminoacid substitutions thereof. In some embodiments, the one or more aminoacid substitution is A12T/H18L/M43V/F59L/E77K/P109S/I118T,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N1445/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R, K89R/D90K/A91G/F92Y/K93R, or A91G.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of CD28and the ectodomain of PD-L1 with increased affinity compared to theunmodified CD80 polypeptide. In some embodiments, the one or more aminoacid substitutions corresponds to position(s) 12, 18, 36, 40, 43, 59,77, 88, 89, 90, 91, 92, 93, 109, 118, 122, 177 with reference tonumbering of SEQ ID NO: 28. In some embodiments, the one or more aminoacid substitution is selected from the group consisting of A12T, H18L,K36G, L40M, M43V, F59L, E77K, E88D, K89R, D90K, A91G, F92Y, K93R, P109S,I118T, N112S, N177S, and conservative amino acid substitutions thereof.In some embodiments, the one or more amino acid substitution isA12T/H18L/M43V/F59L/E77K/P109S/I118T, K36G, K36G/L40M,E88D/K89R/D90K/A91G/F92Y/K93R, K89R/D90K/A91G/F92Y/K93R, orK89R/D90K/A91G/F92Y/K93R/N122S/N177S.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of CTLA-4with increased affinity compared to the unmodified CD80 polypeptide. Insome embodiments, the increased affinity to the ectodomain is increasedmore than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or60-fold compared to the binding affinity of the unmodified CD80 for theectodomain. In some of any such embodiments, the variant polypeptidespecifically binds to the ectodomain of CTLA-4 with increasedselectivity compared to the binding of the unmodified CD80 for theectodomain. In some cases, the increased selectivity includes a greaterratio for binding CTLA-4 versus CD28 or PD-L1 compared to the ratio ofbinding of the unmodified CD80 polypeptide for CTLA-4 versus CD28 orPD-L1. In some embodiments, the ratio is greater by at least or at leastabout 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold. 5-fold, 10-fold, 15-fold,20-fold, 30-fold, 40-fold, 50-fold or more.

In some embodiments, the one or more amino acid substitutionscorresponds to position(s) 4, 29, 31, 36, 40, 41, 52, 67, 68, 70, 87,88, 89, 90, 91, 92, 93, 107, 109, 110, 118, 120, 130, 144, or 169 withreference to numbering of SEQ ID NO: 28. In some embodiments, the one ormore amino acid substitution is selected from the group consisting ofV4M, R29H, Y31H, K36G, L40M, T41G, E52G, I67T, V68A, L70Q, Y87N, E88D,E88G, K89E, K89R, D90K, D90N, A91G, F92Y, K93R, D107N, P109S, T110A,I118V, T120S, T130A, N144D, and K169E and conservative amino acidsubstitutions thereof. In some embodiments, the one or more amino acidsubstitution is V4M/L70Q/A91G/T120S/T130A,V4M/L70Q/A91G/I118V/T120S/T130A/K169E,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,E52G/L70Q/A91G/T120S/T130A,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E, I67T/L70Q/A91G/T120S,I67T/L70Q/A91G/I118V/T120S, V68A/T110A, L70Q/A91G, L70Q/A91G/N144D,L70Q/A91G/T120S/T130A, L70Q/A91G/I118V/T120S/T130A/K169E,L70Q/A91G/T130A, K89R/D90K/A91G/F92Y/K93R,E88D/K89R/D90K/A91G/F92Y/K93R, A91G/I118V/T120S/T130A, orA91G/T120S/T130A.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of CD28and the ectodomain of CTLA-4 with increased affinity compared to theunmodified CD80 polypeptide. In some embodiments, the one or more aminoacid substitutions correspond(s) to position(s) 36, 40, 52, 70, 88, 89,90, 91, 92, 93, 107, 118, 120, 130, 144, or 169 of SEQ ID NO: 28. Insome embodiments, the one or more amino acid substitution is selectedfrom the group consisting of K36G, L40M, E52G, L70Q, E88D, K89R, D90K,A91G, F92Y, K93R, D107N, I118V, T120S, T130A, N144D, and K169E, andconservative amino acid substitutions thereof. In some embodiments, theone or more amino acid substitution is K36G, K36G/L40M,E52G/L70Q/A91G/T120S/T130A,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E, L70Q/A91G,L70Q/A91G/N144D, L70Q/A91G/T120S/T130A,L70Q/A91G/I118V/T120S/T130A/K169E, E88D/K89R/D90K/A91G/F92Y/K93R, orK89R/D90K/A91G/F92Y/K93R.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of PD-L1and the ectodomain of CTLA-4 with increased affinity compared to theunmodified CD80 polypeptide. In some embodiments, the one or more aminoacid substitutions corresponds to position(s) 29, 31, 36, 40, 41, 67,70, 87, 88, 89, 90, 91, 92, 93, 109, 118, 120, 122, or 178 of SEQ ID NO:28. In some embodiments, the one or more amino acid substitution isselected from the group consisting of R29H, Y31H, K36G, L40M, T41G,I67T, L70Q, Y87N, E88D, E88G, K89E, K89R, D90N, D90K, A91G, F92Y, K93R,P109S, I118V, T120S, and conservative amino acid substitutions thereof.In some embodiments, the one or more amino acid substitution isR29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R, or K89R/D90K/A91G/F92Y/K93R.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of CD28,the ectodomain of PD-L1, and the ectodomain of CTLA-4 with increasedaffinity compared to the unmodified CD80 polypeptide. In someembodiments, the one or more amino acid substitutions corresponds toposition(s) 36, 40, 88, 89, 90, 91, 92, or 93 of SEQ ID NO: 28. In someembodiments, the one or more amino acid substitution is selected fromthe group consisting of K36G, L40M, E88D, K89R, D90K, A91G, F92Y, K93R,and conservative amino acid substitutions thereof. In some embodiments,the one or more amino acid substitution is K36G, K36G/L40M,E88D/K89R/D90K/A91G/F92Y/K93R, or K89R/D90K/A91G/F92Y/K93R.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of CD28 orthe ectodomain of PD-L1 with increased affinity compared to theunmodified CD80 polypeptide, and the variant CD80 polypeptidespecifically binds to the ectodomain of CTLA-4 with decreased affinitycompared to the unmodified CD80 polypeptide. In some embodiments, theone or more amino acid substitutions corresponds to position(s) 29, 31,33, 36, 38, 41, 42, 43, 47, 63, 67, 70, 81, 85, 87, 88, 89, 90, 91, 92,93, 94, 109, 114, 118, 120, 127, 130, 144, 148, or 149 of SEQ ID NO: 28.In some embodiments, the one or more amino acid substitution is selectedfrom the group consisting of R29D, R29H, Y31H, Y31L, Q33H, K36G, M38I,T41A, T41G, M42T, M43R, M47T, N63S, I67T, L70Q, E81A, E81V, L85R, Y87N,E88G, K89E, K89N, D90N, A91G, A91T, F92P, K93V, R94L, P109S, S114T,I118T, I118V, T120S, I127T, T130A, N144S, L148S, and N149S, andconservative amino acid substitutions thereof. In some embodiments, theone or more amino acid substitution is N63S/L70Q/A91G/T120S/T130A,N63S/L70Q/A91G/S114T/I118V/T120S/T130A, orL70Q/Y87N/A91G/T120S/I127T/T130A.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of CD28with increased affinity compared to the unmodified CD80 polypeptide, andthe variant CD80 polypeptide specifically binds to the ectodomain ofCTLA-4 with decreased affinity compared to the unmodified CD80polypeptide. In some embodiments, the one or more amino acidsubstitutions corresponds to position(s) 63, 70, 81, 87, 91, 114, 118,120, 127, or 130 of SEQ ID NO: 28. In some embodiments, the one or moreamino acid substitution is selected from the group consisting of N63S,L70Q, E81A, Y87N, A91G, S114T, I118V, T120S, I127T, and T130A, andconservative amino acid substitutions thereof. In some embodiments, theone or more amino acid substitution isR29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S,N63S/L70Q/A91G/T120S/T130A, N63S/L70Q/A91G/S114T/I118V/T120S/T130A,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S, orL70Q/Y87N/A91G/T120S/I127T/T130A.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of PD-L1with increased affinity compared to the unmodified CD80 polypeptide, andthe variant CD80 polypeptide specifically binds to the ectodomain ofCTLA-4 with decreased affinity compared to the unmodified CD80polypeptide. In some embodiments, the one or more amino acidsubstitutions corresponds to position(s) 29, 31, 33, 36, 38, 41, 42, 43,47, 67, 70, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 109, 118, 120, 144,148, 149 of SEQ ID NO: 28. In some embodiments, the one or more aminoacid substitution is selected from the group consisting of R29D, R29H,Y31H, Y31L, Q33H, K36G, M38I, T41A, T41G, M42T, M43R, M47T, I67T, L70Q,E81V, L85R, Y87N, E88G, K89E, K89N, D90N, A91G, A91T, F92P, K93V, R94L,P109S, I118T, I118V, T120S, N144S, L148S, and N149S, and conservativeamino acid substitutions thereof. In some embodiments, the one or moreamino acid substitution isR29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S,I67T/L70Q/A91G/I118V/T120S, or I67T/L70Q/A91G/T120S.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide specifically binds to the ectodomain of CD28and the ectodomain of PD-L1 with increased affinity compared to theunmodified CD80 polypeptide, and the variant CD80 polypeptidespecifically binds to the ectodomain of CTLA-4 with decreased affinitycompared to the unmodified CD80 polypeptide. In some embodiments, theone or more amino acid substitutions correspond(s) to position(s) of 70,81, 87, 91, or 120 of SEQ ID NO: 28. In some embodiments, the one ormore amino acid substitution is selected from the group consisting ofL70Q, Y87N, A91G, and T120S, and conservative amino acid substitutionsthereof.

In some embodiments of any one of the variant CD80 polypeptides, theCD28 is a human CD28. In some embodiments, the PD-L1 is a human PD-L1.In some embodiments, the CTLA-4 is a human CTLA-4.

In some embodiments of any one of the variant CD80 polypeptides, thebinding activity is altered (increased or decreased) more than 1.2-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold compared to theunmodified CD80 polypeptide.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide is a soluble protein.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide is linked to a multimerization domain. In someembodiments, the variant CD80 polypeptide is a multimeric polypeptide,optionally a dimeric polypeptide, comprising a first variant CD80polypeptide linked to a multimerization domain and a second variant CD80polypeptide linked to a multimerization domain. In some embodiments, thefirst variant CD80 polypeptide and the second variant CD80 polypeptideare the same or different. In some embodiments, the multimerizationdomain is an Fc domain or a variant thereof with reduced effectorfunction.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 polypeptide is linked to a moiety that increases biologicalhalf-life of the polypeptide. In some embodiments, the variant CD80polypeptide is linked to an Fc domain or a variant thereof with reducedeffector function. In some embodiments, the Fc domain is mammalian,optionally human; or the variant Fc domain contains one or more aminoacid modifications compared to an unmodified Fc domain that ismammalian, optionally human. In some embodiments, the Fc domain orvariant thereof contains the sequence of amino acids set forth in SEQ IDNO:226 or SEQ ID NO:227 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% sequence identity to SEQ ID NO:226 or SEQ ID NO:227. In someembodiments, the variant CD80 polypeptide is linked indirectly via alinker.

In some embodiments of any one of the variant CD80 polypeptides that isa transmembrane immunomodulatory protein, the variant CD80 polypeptidefurther contains a transmembrane domain linked to the extracellulardomain (ECD) or specific binding fragment thereof of the variant CD80polypeptide. In some embodiments, the transmembrane domain contains thesequence of amino acids set forth as residues 243-263 of SEQ ID NO: 1 ora functional variant thereof that exhibits at least 85% sequenceidentity to residues 243-263 of SEQ ID NO: 1. In some embodiments, thevariant CD80 polypeptide further contains a cytoplasmic signaling domainlinked to the transmembrane domain. In some embodiments, the cytoplasmicsignaling domain contains the sequence of amino acids set forth asresidues 264-288 of SEQ ID NO: 1 or a functional variant thereof thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% sequence identity to residues 264-288 of SEQ ID NO:1.

In some embodiments of any one of the variant CD80 polypeptides, thevariant CD80 increases IFN-gamma (interferon-gamma) expression relativeto the unmodified CD80 in an in vitro primary T-cell assay. In someembodiments of any one of the variant CD80 polypeptides, the variantCD80 decreases IFN-gamma (interferon-gamma) expression relative to theunmodified CD80 in an in vitro primary T-cell assay. In some embodimentsof any one of the variant CD80 polypeptides, the variant CD80polypeptide is deglycosylated.

In some embodiments, provided herein is an immunomodulatory polypeptidecomprising the variant CD80 according to any one of the embodimentsdescribed herein linked to a second polypeptide comprising animmunoglobulin superfamily (IgSF) domain. In some embodiments, the IgSFdomain is affinity modified and exhibits altered binding to one or moreof its cognate binding partner(s) compared to the unmodified orwild-type IgSF domain. In some embodiments, the IgSF domain exhibitsincreased binding to one or more of its cognate binding partner(s)compared to the unmodified or wild-type IgSF domain. In someembodiments, the variant CD80 is a first variant CD80 and the IgSFdomain of the second polypeptide is an IgSF domain from a second variantCD80 according to any one of the embodiments described herein, whereinthe first and second variant CD80 are the same or different. In someembodiments, the variant CD80 polypeptide is capable of specificallybinding to CD28, PD-L1, or CTLA-4, and the IgSF domain of the secondpolypeptide is capable of binding to a cognate binding partner otherthan one specifically bound by the variant CD80 polypeptide. In someembodiments, the variant CD80 polypeptide is capable of specificallybinding to CD28 or PD-L1, and the IgSF domain is capable of binding to acognate binding partner other than one specifically bound by the variantCD80 polypeptide. In some embodiments, the variant CD80 polypeptide iscapable of specifically binding to CD28 or CTLA-4, and the IgSF domainis capable of binding to a cognate binding partner other than onespecifically bound by the variant CD80 polypeptide. In some embodiments,the variant CD80 polypeptide is capable of specifically binding toCTLA-4 or PD-L1 and the IgSF domain is capable of binding to a cognatebinding partner other than one specifically bound by the variant CD80polypeptide. In some embodiments, the IgSF domain is from a member ofthe B7 family. In some embodiments, the IgSF domain is atumor-localizing moiety that binds to a ligand expressed on a tumor. Insome embodiments, the ligand is B7H6. In some embodiments, the IgSFdomain is from NKp30. In some embodiments, the IgSF domain is affinitymodified and exhibits increased binding to one or more of its cognatebinding partner(s) compared to the unmodified or wild-type IgSF domain.In some embodiments, the IgSF domain is or contains an IgV domain. Insome embodiments, the variant CD80 polypeptide is or contains an IgVdomain.

In some embodiments according to any one of the immunomodulatoryproteins, the immunomodulatory protein contains a multimerization domainlinked to one or both of the variant CD80 polypeptide and the secondpolypeptide comprising the IgSF domain. In some embodiments, themultimerization domain is an Fc domain or a variant thereof with reducedeffector function. In some embodiments, the immunomodulatory protein isdimeric. In some embodiments, the immunomodulatory protein ishomodimeric. In some cases, the immunomodulatory protein isheterodimeric.

In some embodiments, provided herein is a conjugate comprising a variantCD80 according to any one of the embodiments described herein or animmunomodulatory polypeptide according to any one of the embodimentsdescribed herein linked to a moiety. In some embodiments, the moiety isa targeting moiety that specifically binds to a molecule on the surfaceof a cell. In some embodiments, the targeting moiety specifically bindsto a molecule on the surface of an immune cell. In some embodiments, theimmune cell is an antigen presenting cell or a lymphocyte. In someembodiments, the targeting moiety is a tumor-localizing moiety thatbinds to a molecule on the surface of a tumor. In some embodiments, themoiety is a protein, a peptide, nucleic acid, small molecule ornanoparticle. In some embodiments, the moiety is an antibody orantigen-binding fragment. In some of any such embodiments, the conjugateis divalent, tetravalent, hexavalent or octavalent.

In some embodiments, provided herein is a nucleic acid molecule encodinga variant CD80 according to any one of the embodiments described hereinor an immunomodulatory polypeptide according to any one of theembodiments described herein. In some embodiments, the nucleic acidmolecule is a synthetic nucleic acid. In some embodiments, the nucleicacid is cDNA.

In some embodiments, provided herein is a vector comprising the nucleicacid of any one of the embodiments described herein. In someembodiments, the vector is an expression vector.

In some embodiments, the vector is a mammalian vector or a viral vector.

In some embodiments, provided herein is a cell comprising the vectoraccording to any one of the embodiments described herein. In someembodiments, the cell is a mammalian cell. In some embodiments, the cellis a human cell.

In some embodiments, provided herein is a method of producing a variantCD80 polypeptide or an immunomodulatory protein, comprising introducingthe nucleic acid molecule according to any one of the embodimentsdescribed herein or vector according to any one of the embodimentsdescribed herein into a host cell under conditions to express theprotein in the cell. In some embodiments, the method further includesisolating or purifying the variant CD80 polypeptide or immunomodulatoryprotein from the cell.

In some embodiments, provided herein is a method of engineering a cellexpressing a variant CD80 polypeptide, comprising introducing a nucleicacid molecule encoding the variant CD80 polypeptide according to any oneof the embodiments described herein into a host cell under conditions inwhich the polypeptide is expressed in the cell.

In some embodiments, provided herein is an engineered cell, expressingthe variant CD80 polypeptide according to any one of the embodimentsdescribed herein, the immunomodulatory protein according to any one ofthe embodiments described herein, the nucleic acid molecule according toany one of the embodiments described herein, or the vector according toany one of the embodiments described herein. In some embodiments, thevariant CD80 polypeptide or immunomodulatory polypeptide contains asignal peptide. In some aspects, the variant CD80 polypeptide orimmunomodulatory polypeptide does not contain a transmembrane domainand/or is not expressed on the surface of the cell. In some embodiments,the variant CD80 polypeptide or immunomodulatory polypeptide is secretedfrom the engineered cell.

In some embodiments, the engineered cell contains a variant CD80polypeptide that contains a transmembrane domain and/or is thetransmembrane immunomodulatory protein according to any one of theembodiments described herein. In some embodiments, the variant CD80polypeptide is expressed on the surface of the cell.

In some embodiments, the engineered cell is an immune cell. In someembodiments, the immune cell is an antigen presenting cell (APC) or alymphocyte. In some embodiments, the engineered cell is a primary cell.In some embodiments, the engineered cell is a mammalian cell. In someembodiments, the engineered cell is a human cell. In some embodiments,the lymphocyte is a T cell. In some embodiments, the APC is anartificial APC.

In some of any such embodiments, the engineered cell further contains achimeric antigen receptor (CAR) or an engineered T-cell receptor.

Also provided is an infectious agent, comprising a nucleic acid moleculeencoding a variant CD80 polypeptide according to any one of theembodiments described herein or an immunomodulatory polypeptideaccording to any one of the embodiments described herein. In someinstances, the encoded variant CD80 polypeptide or immunomodulatorypolypeptide does not contain a transmembrane domain and/or is notexpressed on the surface of a cell in which it is expressed. In someembodiments, the encoded variant CD80 polypeptide or immunomodulatorypolypeptide is secreted from a cell in which it is expressed. In someaspects, the encoded variant CD80 polypeptide contains a transmembranedomain. In some aspects, the encoded variant CD80 polypeptide isexpressed on the surface of a cell in which it is expressed.

In some of any such embodiments, the infectious agent is a bacterium ora virus. In some cases, the virus is an oncolytic virus. In someembodiments, the oncolytic virus is an adenoviruses, adeno-associatedviruses, herpes viruses, Herpes Simplex Virus, Vesticular Stomaticvirus, Reovirus, Newcastle Disease virus, parvovirus, measles virus,vesticular stomatitis virus (VSV), Coxsackie virus or a Vaccinia virus.In some embodiments, the virus specifically targets dendritic cells(DCs) and/or is dendritic cell-tropic. In some instances, the virus is alentiviral vector that is pseudotyped with a modified Sindbis virusenvelope product.

In some of any such embodiments, the infectious agent further contains anucleic acid molecule encoding a further gene product that results indeath of a target cell or that can augment or boost an immune response.In some embodiments, the further gene product is selected from ananticancer agent, anti-metastatic agent, an antiangiogenic agent, animmunomodulatory molecule, an immune checkpoint inhibitor, an antibody,a cytokine, a growth factor, an antigen, a cytotoxic gene product, apro-apoptotic gene product, an anti-apoptotic gene product, a cellmatrix degradative gene, genes for tissue regeneration or areprogramming human somatic cells to pluripotency.

In some embodiments, provided herein is a pharmaceutical composition,comprising the variant CD80 polypeptide according to any one of theembodiments described herein, an immunomodulatory protein according toany one of the embodiments described herein, a conjugate according toany one of the embodiments described herein, or an engineered cellaccording to any one of the embodiments described herein. In someembodiments, the pharmaceutical composition further includes apharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition is sterile.

In some embodiments, provided herein is an article of manufacturecomprising the pharmaceutical composition according to any one of theembodiments described herein in a vial. In some embodiments, the vial issealed.

In some embodiments, provided herein is a kit comprising thepharmaceutical composition according to any one of the embodimentsdescribed herein and instructions for use. In some embodiments, providedherein is a kit comprising the article of manufacture according to anyone of the embodiments described herein and instructions for use.

In some embodiments, provided herein is a method of modulating an immuneresponse in a subject, comprising administering the pharmaceuticalcomposition according to any one of the embodiments described herein tothe subject. In some embodiments, the method includes administering theengineered cells according to any one of the embodiments describedherein. In some embodiments, the engineered cells are autologous to thesubject. In some embodiments, the engineered cells are allogenic to thesubject

In some embodiments, modulating the immune response treats a disease orcondition in the subject. In some embodiments, the immune response isincreased. In some embodiments, an immunomodulatory protein or conjugatecomprising a variant CD80 polypeptide linked to a tumor-localizingmoiety is administered to the subject. In some cases, thetumor-localizing moiety is or contains a binding molecule thatrecognizes a tumor antigen. In some instances, the binding moleculecontains an antibody or an antigen-binding fragment thereof or containsa wild-type IgSF domain or variant thereof.

In some embodiments, a pharmaceutical composition comprising theimmunomodulatory protein according to any one of the embodimentsdescribed herein or the conjugate according to any one of theembodiments described herein is administered to the subject. In someembodiments, an engineered cell comprising a variant CD80 polypeptidethat is a transmembrane immunomodulatory protein is administered to thesubject and/or the engineered cell according to any one of theembodiments described herein is administered to the subject.

In some embodiments, an infectious agent encoding a variant CD80polypeptide that is a transmembrane immunomodulatory protein isadministered to the subject, optionally under conditions in which theinfectious agent infects a tumor cell or immune cell and thetransmembrane immunomodulatory protein is expressed on the surface ofthe infected cell. In some aspects, the transmembrane immunomodulatoryprotein is a transmembrane immunomodulatory protein according to any oneof the embodiments described herein.

In some embodiments, the disease or condition is a tumor or cancer. Insome embodiments, the disease or condition is selected from melanoma,lung cancer, bladder cancer, hematological malignancy, liver cancer,brain cancer, renal cancer, breast cancer, pancreatic cancer, colorectalcancer, spleen cancer, prostate cancer, testicular cancer, ovariancancer, uterine cancer, gastric carcinoma, a musculoskeletal cancer, ahead and neck cancer, a gastrointestinal cancer, a germ cell cancer, oran endocrine and neuroendocrine cancer.

In some embodiments, the immune response is decreased by the providedmethods of modulating the immune response.

In some embodiments, a variant CD80 polypeptide or immunomodulatoryprotein that is soluble is administered to the subject. In some cases,the soluble polypeptide or immunomodulatory protein is an Fc fusionprotein. In some of any such embodiments, a pharmaceutical compositioncomprising a variant CD80 polypeptide according to any one of theembodiments described herein, or the immunomodulatory protein accordingto any one of the embodiments described herein is administered to thesubject. In some embodiments, an engineered cell comprising a secretablevariant CD80 polypeptide is administered to the subject. In someembodiments, an engineered cell according to any one of the embodimentsdescribed herein is administered to the subject.

In some embodiments, an infectious agent encoding a variant CD80polypeptide that is a secretable immunomodulatory protein isadministered to the subject, optionally under conditions in which theinfectious agent infects a tumor cell or immune cell and the secretableimmunomodulatory protein is secreted from the infected cell.

In some of any such embodiments, the disease or condition is aninflammatory or autoimmune disease or condition. In some embodiments,the disease or condition is an Antineutrophil cytoplasmic antibodies(ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease,transplantation, a Rheumatic disease, an inflammatory gastrointestinaldisease, an inflammatory eye disease, an inflammatory neurologicaldisease, an inflammatory pulmonary disease, an inflammatory endocrinedisease, or an autoimmune hematological disease. In some embodiments,the disease or condition is selected from inflammatory bowel disease,transplant, Crohn's disease, ulcerative colitis, multiple sclerosis,asthma, rheumatoid arthritis, or psoriasis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C depicts various formats of the provided variant IgSF domainmolecules. FIG. 1A depicts soluble molecules, including: (1) a variantIgSF domain (vIgD), e.g. variant CD80, fused to an Fc chain; (2) a stackmolecule containing a first variant IgSF domain (first vIgD), e.g.variant CD80, and a second IgSF domain, such as a second variant IgSFdomain (second vIgD); (3) a tumor targeting IgSF molecule containing afirst variant IgSF domain (vIgD), e.g. variant CD80, and an IgSF domainthat targets to a tumor antigen, such as an NkP30 IgSF domain; and (4) avariant IgSF domain (vIgD), e.g. variant CD80, linked to an antibody(V-Mab). FIG. 1B depicts a transmembrane immunomodulatory protein (TIP)containing a variant IgSF domain (vIgD) expressed on the surface of acell. In an exemplary embodiment, the cognate binding partner of thetransmembrane bound vIgD is a costimulatory receptor, and the TIPcontaining the vIgD (e.g. CD80 vIgD) agonizes the costimulatory receptorsuch that the TIP induces a positive signal in the cell expressing thecostimulatory receptor. FIG. 1C depicts a secreted immunomodulatoryprotein (SIP) in which a variant IgSF domain (vIgD) is secreted from acell, such as a first T cell (e.g. CAR T cell). In an exemplaryembodiment, the cognate binding partner of the secreted vIgD is anactivating receptor, e.g. CD28, which can be expressed on the first cell(e.g. T cell) and/or on a second cell (e.g. T cell; either endogenous orengineered, such as a CART cell). Upon binding of the SIP with itscognate binding partner, signaling via the activating receptor isblocked. In all cases, the vIgD can be a V-domain (IgV) only, thecombination of the V-domain (IgV) and C-domain (IgC), including theentire extracellular domain (ECD), or any combination of Ig domains ofthe IgSF superfamily member.

FIG. 2 depicts an exemplary schematic of the activity of a variant IgSFdomain (vIgD) fused to an Fc (vIgD-Fc) in which the vIgD is a variant ofan IgSF domain of CD80. As shown, a soluble vIgD of CD80 interacts withits cognate binding partners to block interactions of CD28,respectively, thereby blocking costimulation by the CD28 costimulatoryreceptors.

FIG. 3 depicts an exemplary schematic of a stack molecule for localizingthe variant IgSF domain (vIgD) to a tumor cell. In this format, thestack molecule contains a first variant IgSF domain (first vIgD) and asecond IgSF domain (e.g. a second vIgD) in which the second IgSF domain(e.g a second vIgD) is a tumor-targeted IgSF domain that binds to atumor antigen. An exemplary tumor-targeted IgSF domain is an IgSF domainof NkP30, which binds to the tumor antigen B7-H6. In this depiction, thevIgD is a variant of an IgSF domain of CD80. As shown, binding oftumor-targeted IgSF domain to the surface of the tumor cell localizesthe first vIgD on the tumor cell surface where it can interact with oneor more of its cognate binding partner (e.g. CD28) expressed on thesurface of an adjacent immune cell (e.g. T cell) to stimulate thecostimulatory receptor.

FIG. 4 depicts various exemplary configurations of a stack moleculecontaining a first variant IgSF domain (first vIgD) and a second IgSFdomain, such as a second variant IgSF domain (second vIgD). As shown,the first vIgD and second IgSF domain are independently linked, directlyor indirectly, to the N- or C-terminus of an Fc subunit. For generatinga homodimeric Fc molecule, the Fc subunit is one that is capable offorming a homodimer with a matched Fc subunit by co-expression of theindividual Fc subunits in a cell. For generating a heterodimeric Fcmolecule, the individual Fc subunits contain mutations (e.g.“knob-into-hole” mutations in the CH3 domain), such that formation ofthe heterodimer is favored compared to homodimers when the individual Fcsubunits are co-expressed in a cell.

FIG. 5 depicts an exemplary schematic of the activity of a variant IgSFdomain (vIgD) conjugated to an antibody (V-Mab) in which the antibody(e.g. anti-HER2 antibody) binds to an antigen on the surface of thetumor cell. In this depiction, the vIgD is a variant of an IgSF domainof CD80. As shown, binding of the antibody to the surface of the tumorcell localizes the vIgD on the tumor cell surface where it can interactwith one or more of its cognate binding partners expressed on thesurface of an adjacent immune cell (e.g. T cell) to agonize receptorsignaling. In an exemplary embodiment as shown, the variant IgSF domain(vIgD) is a variant of an IgSF domain of CD80. Binding of the CD80 vIgDto CD28 costimulatory receptors provides an agonist or costimulatorysignal.

FIG. 6A-6C depicts various exemplary configurations of a variant IgSFdomain (vIgD) conjugated to an antibody (V-Mab). FIG. 6A shows variousconfigurations in which a vIgD is linked, directly or indirectly, to theN- and/or C-terminus of the light chain of an antibody. FIG. 6B showsvarious configurations in which a vIgD is linked, directly orindirectly, to the N- and/or C-terminus of the heavy chain of anantibody. FIG. 6C depicts the resulting V-Mab configurations when alight chain of FIG. 6A and a heavy chain of FIG. 6B are co-expressed ina cell.

FIG. 7A depicts results of a competition binding assay for binding ofbiotinylated recombinant CD28 Fc fusion protein (rCD28.Fc) toimmobilized variant CD80 A91G ECD-Fc fusion molecule in the presence ofunlabeled recombinant human PD-L1-his, human CTLA-4-his orhuman-PD-L2-Fc fusion protein.

FIG. 7B depicts results of a competition binding assay for binding ofbiotinylated recombinant human PD-L1-his monomeric protein toimmobilized variant CD80 A91G ECD-Fc fusion molecule in the presence ofunlabeled recombinant human rCD28.Fc, human CTLA-4.Fc or human PD-L2.Fc.

FIG. 8 depicts impedance results reflecting cytotoxic killing activityof cells engineered with an anti-CD19 chimeric antigen receptor (CAR)alone or with an exemplary transmembrane immunomodulatory TIP (CD80-TIPor ICOSL-TIP) or the corresponding CD80 or ICOSL wild-type transmembraneprotein following co-culture with target antigen-expressing cells.Impedance was assessed using the Acea Real-Time Cell Analyzer (RTCA),which measures the impedance variations in the culture media of a96-well microelectronic plate (E-plate).

DETAILED DESCRIPTION

Provided herein are immunomodulatory proteins that are or comprisevariants or mutants of CD80 and specific binding fragments thereof thatexhibit altered binding activity or affinity to at least one targetligand cognate binding partner (also called counter-structure ligandprotein). In some embodiments, the variant CD80 polypeptides contain oneor more amino acid modifications (e.g., amino acid substitutions,deletions, or additions) compared to an unmodified or wild-type CD80polypeptide. In some embodiments, the variant CD80 polypeptides containone or more amino acid modifications (e.g., substitutions) compared toan unmodified or wild-type CD80 polypeptide. In some embodiments, theone or more amino acid modifications (e.g., substitutions) are in anIgSF domain (e.g. IgV) of an unmodified or wild-type CD80 polypeptide.In some embodiments, the altered binding activity or affinity, such asincreased or decreased binding activity or affinity, is for at least oneof the cognate binding partner proteins CD28, PD-L1, or CTLA-4. In someembodiments, the variant CD80 polypeptides exhibit altered, such asincreased or decreased, binding activity or affinity to one or more ofCD28, PD-L1, or CTLA-4 compared to the unmodified or wild-type CD80 notcontaining the one or more modifications. In some embodiments, theimmunomodulatory proteins are soluble. In some embodiments, theimmunomodulatory proteins are transmembrane immunomodulatory proteinscapable of being expressed on the surface of cells. In some embodiments,also provided herein are one or more other immunomodulatory proteinsthat are conjugates or fusions containing a variant CD80 polypeptideprovided herein and one or more other moiety or polypeptide.

In some embodiments, the variant CD80 polypeptides exhibit increasedbinding affinity to one or more of CD28, PD-L1, or CTLA-4 compared tothe unmodified or wild-type CD80 not containing the one or moremodifications. In some embodiments, the variant CD80 polypeptidesexhibit decreased binding affinity to one or more of CD28, PD-L1, orCTLA-4 compared to the unmodified or wild-type CD80 not containing theone or more modifications. In some embodiments, the variant CD80polypeptides exhibit increased binding affinity to one or more of CD28,PD-L1, or CTLA-4, and decreased binding affinity to another one or moreof CD28, PD-L1, or CTLA-4 compared to the unmodified or wild-type CD80not containing the one or more modifications.

In some embodiments, also provided herein are one or more otherimmunomodulatory proteins that are conjugates or fusions containing avariant CD80 polypeptide provided herein and one or more other moiety orpolypeptide. In some embodiments, the variant CD80 polypeptides andimmunomodulatory proteins modulate an immunological immune response,such an increase or decrease an immune response. In some embodiments,the variant CD80 polypeptides and immunomodulatory proteins providedherein can be used for the treatment of diseases or conditions that areassociated with a dysregulated immune response.

In some embodiments, the provided variant CD80 polypeptides modulate Tcell activation via interactions with costimulatory signaling molecules.In general, antigen specific T-cell activation generally requires twodistinct signals. The first signal is provided by the interaction of theT-cell receptor (TCR) with major histocompatibility complex (MHC)associated antigens present on antigen presenting cells (APCs). Thesecond signal is costimulatory to TCR engagement and necessary to avoidT-cell apoptosis or anergy.

In some embodiments, under normal physiological conditions, the Tcell-mediated immune response is initiated by antigen recognition by theT cell receptor (TCR) and is regulated by a balance of co-stimulatoryand inhibitory signals (e.g., immune checkpoint proteins). The immunesystem relies on immune checkpoints to prevent autoimmunity (i.e.,self-tolerance) and to protect tissues from excessive damage during animmune response, for example during an attack against a pathogenicinfection. In some cases, however, these immunomodulatory proteins canbe dysregulated in diseases and conditions, including tumors, as amechanism for evading the immune system.

In some embodiments, among known T-cell costimulatory receptors is CD28,which is the T-cell costimulatory receptor for the ligands B7-1 (CD80)and B7-2 (CD86) both of which are present on APCs. These same ligandscan also bind to the inhibitory T-cell receptor CTLA-4 (cytotoxicT-lymphocyte-associated protein 4) with greater affinity than for CD28;the binding to CTLA-4 acts to down-modulate the immune response.Likewise, CD80 is able to bind to programmed death ligand 1 (PD-L1).PD-L1 also is a negative regulator of immune activation and is capableof down-modulating the immune response via interactions with programmeddeath 1 (PD-1) receptor. The binding of CD80 to PD-L1 can block theinteraction between PD-L1 and PD-1, and thereby potentiate or enhancethe immune response. Thus, in some cases, interactions of CD80 with CD28and PD-L1 yield overlapping and complementary effects.

In some embodiments, CD28 and PD-L1 may play complementary roles inmodulating an immune response. Enhancement or suppression of theactivity of these receptors has clinical significance for treatment ofinflammatory and autoimmune disorders, cancer, and viral infections. Insome cases, however, therapies to intervene and alter the costimulatoryeffects of both receptors are constrained by the spatial orientationrequirements as well as size limitations imposed by the confines of theimmunological synapse. In some aspects, existing therapeutic drugs,including antibody drugs, may not be able to interact simultaneouslywith the multiple target proteins involved in modulating theseinteractions. In addition, in some cases, existing therapeutic drugs mayonly have the ability to antagonize, but not agonize, an immuneresponse. Additionally, pharmacokinetic differences between drugs thatindependently target one or the other of these two receptors can createdifficulties in properly maintaining a desired blood concentration ofsuch drug combinations throughout the course of treatment.

In some embodiments, the provided variant CD80 polypeptides orimmunomodulatory proteins modulate (e.g. increase or decrease)immunological activity induced or associated with costimulatoryreceptors CD28 or PD-L1 and, in some cases, CTLA-4. Thus, in someembodiments, the provided polypeptides overcome these constraints byproviding variants CD80 with independent binding affinities to both CD28and PD-L1, and, in some cases, CTLA-4, thereby agonizing or antagonizingthe complementary effects of costimulation by receptors. Methods ofmaking and using these variants of CD80 are also provided.

All publications, including patents, patent applications scientificarticles and databases, mentioned in this specification are hereinincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, including patent, patentapplication, scientific article or database, were specifically andindividually indicated to be incorporated by reference. If a definitionset forth herein is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

I. DEFINITIONS

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood by one of ordinaryskill in the art to which the claimed subject matter pertains. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.

The terms used throughout this specification are defined as followsunless otherwise limited in specific instances. As used in thespecification and the appended claims, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise. Unless defined otherwise, all technical and scientific terms,acronyms, and abbreviations used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which theinvention pertains. Unless indicated otherwise, abbreviations andsymbols for chemical and biochemical names is per IUPAC-IUBnomenclature. Unless indicated otherwise, all numerical ranges areinclusive of the values defining the range as well as all integer valuesin-between.

The term “affinity modified” as used in the context of an immunoglobulinsuperfamily domain, means a mammalian immunoglobulin superfamily (IgSF)domain having an altered amino acid sequence (relative to thecorresponding wild-type parental or unmodified IgSF domain) such that ithas an increased or decreased binding affinity or avidity to at leastone of its cognate binding partners (alternatively “counter-structures”)compared to the parental wild-type or unmodified (i.e., non-affinitymodified) IgSF control domain. Included in this context is an affinitymodified CD80 IgSF domain. In some embodiments, the affinity-modifiedIgSF domain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or moreamino acid differences, such as amino acid substitutions, in a wildtypeor unmodified IgSF domain. An increase or decrease in binding affinityor avidity can be determined using well known binding assays such asflow cytometry. Larsen et al., American Journal of Transplantation, Vol5: 443-453 (2005). See also, Linsley et al., Immunity, 1: 7930801(1994). An increase in a protein's binding affinity or avidity to itscognate binding partner(s) is to a value at least 10% greater than thatof the wild-type IgSF domain control and in some embodiments, at least20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000%greater than that of the wild-type IgSF domain control value. A decreasein a protein's binding affinity or avidity to at least one of itscognate binding partner is to a value no greater than 90% of the controlbut no less than 10% of the wild-type IgSF domain control value, and insome embodiments no greater than 80%, 70% 60%, 50%, 40%, 30%, or 20% butno less than 10% of the wild-type IgSF domain control value. Anaffinity-modified protein is altered in primary amino acid sequence bysubstitution, addition, or deletion of amino acid residues. The term“affinity modified IgSF domain” is not be construed as imposing anycondition for any particular starting composition or method by which theaffinity-modified IgSF domain was created. Thus, the affinity modifiedIgSF domains of the present invention are not limited to wild type IgFdomains that are then transformed to an affinity modified IgSF domain byany particular process of affinity modification. An affinity modifiedIgSF domain polypeptide can, for example, be generated starting fromwild type mammalian IgSF domain sequence information, then modeled insilico for binding to its cognate binding partner, and finallyrecombinantly or chemically synthesized to yield the affinity modifiedIgSF domain composition of matter. In but one alternative example, anaffinity modified IgSF domain can be created by site-directedmutagenesis of a wild-type IgSF domain. Thus, affinity modified IgSFdomain denotes a product and not necessarily a product produced by anygiven process. A variety of techniques including recombinant methods,chemical synthesis, or combinations thereof, may be employed.

The term “allogeneic” as used herein means a cell or tissue that isremoved from one organism and then infused or adoptively transferredinto a genetically dissimilar organism of the same species. In someembodiments of the invention, the species is murine or human.

The term “autologous” as used herein means a cell or tissue that isremoved from the same organism to which it is later infused oradoptively transferred. An autologous cell or tissue can be altered by,for example, recombinant DNA methodologies, such that it is no longergenetically identical to the native cell or native tissue which isremoved from the organism. For example, a native autologous T-cell canbe genetically engineered by recombinant DNA techniques to become anautologous engineered cell expressing a transmembrane immunomodulatoryprotein and/or chimeric antigen receptor (CAR), which in some casesinvolves engineering a T-cell or TIL (tumor infiltrating lymphocyte).The engineered cells are then infused into a patient from which thenative T-cell was isolated. In some embodiments, the organism is humanor murine.

The terms “binding affinity,” and “binding avidity” as used herein meansthe specific binding affinity and specific binding avidity,respectively, of a protein for its counter-structure under specificbinding conditions. In biochemical kinetics avidity refers to theaccumulated strength of multiple affinities of individual non-covalentbinding interactions, such as between CD80 and its counter-structuresPD-L1, CD28, and/or CTLA-4. As such, avidity is distinct from affinity,which describes the strength of a single interaction. An increase orattenuation in binding affinity of a variant CD80 containing an affinitymodified CD80 IgSF domain to its counter-structure is determinedrelative to the binding affinity of the unmodified CD80, such as anunmodified CD80 containing the native or wild-type IgSF domain, such asIgV domain. Methods for determining binding affinity or avidity areknown in art. See, for example, Larsen et al., American Journal ofTransplantation, Vol 5: 443-453 (2005). In some embodiments, a variantCD80, such as containing an affinity modified IgSF domain, specificallybinds to CD28, PD-L1 and/or CTLA-4 measured by flow cytometry with abinding affinity that yields a Mean Fluorescence Intensity (MFI) valueat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greaterthan an unmodified CD80 control in a binding assay such as described inExample 6.

The term “biological half-life” refers to the amount of time it takesfor a substance, such as an immunomodulatory polypeptide comprising avariant CD80 polypeptide of the present invention, to lose half of itspharmacologic or physiologic activity or concentration. Biologicalhalf-life can be affected by elimination, excretion, degradation (e.g.,enzymatic) of the substance, or absorption and concentration in certainorgans or tissues of the body. In some embodiments, biological half-lifecan be assessed by determining the time it takes for the blood plasmaconcentration of the substance to reach half its steady state level(“plasma half-life”). Conjugates that can be used to derivatize andincrease the biological half-life of polypeptides of the invention areknown in the art and include, but are not limited to, polyethyleneglycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinantpeptides; see, WO2013130683), human serum albumin (HSA), bovine serumalbumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS),polyglutamic acid (glutamylation).

The term “chimeric antigen receptor” or “CAR” as used herein refers toan artificial (i.e., man-made) transmembrane protein expressed on amammalian cell comprising at least an ectodomain, a transmembrane, andan endodomain. Optionally, the CAR protein includes a “spacer” whichcovalently links the ectodomain to the transmembrane domain. A spacer isoften a polypeptide linking the ectodomain to the transmembrane domainvia peptide bonds. The CAR is typically expressed on a mammalianlymphocyte. In some embodiments, the CAR is expressed on a mammaliancell such as a T-cell or a tumor infiltrating lymphocyte (TIL). A CARexpressed on a T-cell is referred to herein as a “CAR T-cell” or“CAR-T.” In some embodiments the CAR-T is a T helper cell, a cytotoxicT-cell, a natural killer T-cell, a memory T-cell, a regulatory T-cell,or a gamma delta T-cell. When used clinically in, e.g. adoptive celltransfer, a CAR-T with antigen binding specificity to the patient'stumor is typically engineered to express on a native T-cell obtainedfrom the patient. The engineered T-cell expressing the CAR is theninfused back into the patient. The CAR-T is thus often an autologousCAR-T although allogeneic CAR-T are included within the scope of theinvention. The ectodomain of a CAR comprises an antigen binding region,such as an antibody or antigen binding fragment thereof (e.g. scFv),that specifically binds under physiological conditions with a targetantigen, such as a tumor specific antigen Upon specific binding abiochemical chain of events (i.e., signal transduction) results inmodulation of the immunological activity of the CAR-T. Thus, forexample, upon specific binding by the antigen binding region of theCAR-T to its target antigen can lead to changes in the immunologicalactivity of the T-cell activity as reflected by changes in cytotoxicity,proliferation or cytokine production. Signal transduction upon CAR-Tactivation is achieved in some embodiments by the CD3-zeta chain(“CD3-z”) which is involved in signal transduction in native mammalianT-cells. CAR-Ts can further comprise multiple signaling domains such asCD28, 41BB or OX40, to further modulate immunomodulatory response of theT-cell. CD3-z comprises a conserved motif known as an immunoreceptortyrosine-based activation motif (ITAM) which is involved in T-cellreceptor signal transduction.

The term “collectively” or “collective” when used in reference tocytokine production induced by the presence of two or more variant CD80polypeptides in an in vitro assay, means the overall cytokine expressionlevel irrespective of the cytokine production induced by individualvariant CD80 polypeptides. In some embodiments, the cytokine beingassayed is IFN-gamma in an in vitro primary T-cell assay such asdescribed in Example 7.

The term “cognate binding partner” (used interchangeably with“counter-structure”) in reference to a polypeptide, such as in referenceto an IgSF domain of a variant CD80, refers to at least one molecule(typically a native mammalian protein) to which the referencedpolypeptide specifically binds under specific binding conditions. Insome aspects, a variant CD80 containing an affinity modified IgSF domainspecifically binds to the counter-structure of the corresponding nativeor wildtype CD80 but with increased or attenuated affinity. A species ofligand recognized and specifically binding to its cognate receptor underspecific binding conditions is an example of a counter-structure orcognate binding partner of that receptor. A “cognate cell surfacebinding partner” is a cognate binding partner expressed on a mammaliancell surface. A “cell surface molecular species” is a cognate bindingpartner of ligands of the immunological synapse (IS), expressed on andby cells, such as mammalian cells, forming the immunological synapse.

As used herein, “conjugate,” “conjugation” or grammatical variationsthereof refers the joining or linking together of two or more compoundsresulting in the formation of another compound, by any joining orlinking methods known in the art. It can also refer to a compound whichis generated by the joining or linking together two or more compounds.For example, a variant CD80 polypeptide linked directly or indirectly toone or more chemical moieties or polypeptide is an exemplary conjugate.Such conjugates include fusion proteins, those produced by chemicalconjugates and those produced by any other methods.

The term “competitive binding” as used herein means that a protein iscapable of specifically binding to at least two cognate binding partnersbut that specific binding of one cognate binding partner inhibits, suchas prevents or precludes, simultaneous binding of the second cognatebinding partner. Thus, in some cases, it is not possible for a proteinto bind the two cognate binding partners at the same time. Generally,competitive binders contain the same or overlapping binding site forspecific binding but this is not a requirement. In some embodiments,competitive binding causes a measurable inhibition (partial or complete)of specific binding of a protein to one of its cognate binding partnerdue to specific binding of a second cognate binding partner. A varietyof methods are known to quantify competitive binding such as ELISA(enzyme linked immunosorbent assay) assays.

The term “conservative amino acid substitution” as used herein means anamino acid substitution in which an amino acid residue is substituted byanother amino acid residue having a side chain R group with similarchemical properties (e.g., charge or hydrophobicity). Examples of groupsof amino acids that have side chains with similar chemical propertiesinclude 1) aliphatic side chains: glycine, alanine, valine, leucine, andisoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3)amide-containing side chains: asparagine and glutamine; 4) aromatic sidechains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains:lysine, arginine, and histidine; 6) acidic side chains: aspartic acidand glutamic acid; and 7) sulfur-containing side chains: cysteine andmethionine. Conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

The term, “corresponding to” with reference to positions of a protein,such as recitation that nucleotides or amino acid positions “correspondto” nucleotides or amino acid positions in a disclosed sequence, such asset forth in the Sequence Listing, refers to nucleotides or amino acidpositions identified upon alignment with the disclosed sequence based onstructural sequence alignment or using a standard alignment algorithm,such as the GAP algorithm. For example, corresponding residues can bedetermined by alignment of a reference sequence with the sequence ofwild-type CD80 set forth in SEQ ID NO:28 (ECD domain) or set forth inSEQ ID NO:152 or 372 (IgV domain) by structural alignment methods asdescribed herein. By aligning the sequences, one skilled in the art canidentify corresponding residues, for example, using conserved andidentical amino acid residues as guides.

The terms “decrease” or “attenuate” “or suppress” as used herein meansto decrease by a statistically significant amount. A decrease can be atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

The terms “derivatives” or “derivatized” refer to modification of aprotein by covalently linking it, directly or indirectly, to acomposition so as to alter such characteristics as biological half-life,bioavailability, immunogenicity, solubility, toxicity, potency, orefficacy while retaining or enhancing its therapeutic benefit.Derivatives of immunomodulatory polypeptides of the invention are withinthe scope of the invention and can be made by, for example,glycosylation, pegylation, lipidation, or Fc-fusion.

As used herein, domain (typically a sequence of three or more, generally5 or 7 or more amino acids, such as 10 to 200 amino acid residues)refers to a portion of a molecule, such as a protein or encoding nucleicacid, that is structurally and/or functionally distinct from otherportions of the molecule and is identifiable. For example, domainsinclude those portions of a polypeptide chain that can form anindependently folded structure within a protein made up of one or morestructural motifs and/or that is recognized by virtue of a functionalactivity, such as binding activity. A protein can have one, or more thanone, distinct domains. For example, a domain can be identified, definedor distinguished by homology of the primary sequence or structure torelated family members, such as homology to motifs. In another example,a domain can be distinguished by its function, such as an ability tointeract with a biomolecule, such as a cognate binding partner. A domainindependently can exhibit a biological function or activity such thatthe domain independently or fused to another molecule can perform anactivity, such as, for example binding. A domain can be a linearsequence of amino acids or a non-linear sequence of amino acids. Manypolypeptides contain a plurality of domains. Such domains are known, andcan be identified by those of skill in the art. For exemplificationherein, definitions are provided, but it is understood that it is wellwithin the skill in the art to recognize particular domains by name. Ifneeded appropriate software can be employed to identify domains.

The term “ectodomain” as used herein refers to the region of a membraneprotein, such as a transmembrane protein, that lies outside thevesicular membrane. Ectodomains often comprise binding domains thatspecifically bind to ligands or cell surface receptors, such as via abinding domain that specifically binds to the ligand or cell surfacereceptor. The ectodomain of a cellular transmembrane protein isalternately referred to as an extracellular domain.

The terms “effective amount” or “therapeutically effective amount” referto a quantity and/or concentration of a therapeutic composition of theinvention, including a protein composition or cell composition, thatwhen administered ex vivo (by contact with a cell from a patient) or invivo (by administration into a patient) either alone (i.e., as amonotherapy) or in combination with additional therapeutic agents,yields a statistically significant decrease in disease progression as,for example, by ameliorating or eliminating symptoms and/or the cause ofthe disease. An effective amount may be an amount that relieves,lessens, or alleviates at least one symptom or biological response oreffect associated with a disease or disorder, prevents progression ofthe disease or disorder, or improves physical functioning of thepatient. In the case of cell therapy, the effective amount is aneffective dose or number of cells administered to a patient by adoptivecell therapy. In some embodiments the patient is a mammal such as anon-human primate or human patient.

The term “endodomain” as used herein refers to the region found in somemembrane proteins, such as transmembrane proteins, that extends into theinterior space defined by the cell surface membrane. In mammalian cells,the endodomain is the cytoplasmic region of the membrane protein. Incells, the endodomain interacts with intracellular constituents and canbe play a role in signal transduction and thus, in some cases, can be anintracellular signaling domain. The endodomain of a cellulartransmembrane protein is alternately referred to as a cytoplasmicdomain, which, in some cases, can be a cytoplasmic signaling domain.

The terms “enhanced” or “increased” as used herein in the context ofincreasing immunological activity of a mammalian lymphocyte means toincrease one or more activities the lymphocyte. An increased activitycan be one or more of increase cell survival, cell proliferation,cytokine production, or T-cell cytotoxicity, such as by a statisticallysignificant amount. In some embodiments, reference to increasedimmunological activity means to increase interferon gamma (IFN-gamma)production, such as by a statistically significant amount. In someembodiments, the immunological activity can be assessed in a mixedlymphocyte reaction (MLR) assay. Methods of conducting MLR assays areknown in the art. Wang et al., Cancer Immunol Res. 2014 Sep.:2(9):846-56. Other methods of assessing activities of lymphocytes areknown in the art, including any assay as described herein. In someembodiments an enhancement can be an increase of at least 10%, 20%, 30%,40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% greater than a non-zerocontrol value.

The term “engineered cell” as used herein refers to a mammalian cellthat has been genetically modified by human intervention such as byrecombinant DNA methods or viral transduction. In some embodiments, thecell is an immune cell, such as a lymphocyte (e.g. T cell, B cell, NKcell) or an antigen presenting cell (e.g. dendritic cell). The cell canbe a primary cell from a patient or can be a cell line. In someembodiments, an engineered cell of the invention comprises a variantCD80 of the invention engineered to modulate immunological activity of aT-cell expressing CD28, PD-L1 and/or CTLA-4, or an APC expressing PD-L1,to which the variant CD80 polypeptide specifically binds. In someembodiments, the variant 80 is a transmembrane immunomodulatory protein(hereinafter referred to as “TIP”) containing the extracellular domainor a portion thereof containing the IgV domain linked to a transmembranedomain (e.g. a CD80 transmembrane domain) and, optionally, anintracellular signaling domain. In some cases, the TIP is formatted as achimeric receptor containing a heterologous cytoplasmic signaling domainor endodomain. In some embodiments, an engineered cell is capable ofexpressing and secreting a immunomodulatory protein as described herein.Among provided engineered cells also are cells further containing anengineered T-cell receptor (TCR) or chimeric antigen receptor (CAR).

The term “engineered T-cell” as used herein refers to a T-cell such as aT helper cell, cytotoxic T-cell (alternatively, cytotoxic T lymphocyteor CTL), natural killer T-cell, regulatory T-cell, memory T-cell, orgamma delta T-cell, that has been genetically modified by humanintervention such as by recombinant DNA methods or viral transductionmethods. An engineered T-cell comprises a variant CD80 transmembraneimmunomodulatory protein (TIP) of the present invention that isexpressed on the T-cell and is engineered to modulate immunologicalactivity of the engineered T-cell itself, or a mammalian cell to whichthe variant CD80 expressed on the T-cell specifically binds.

The term “engineered T-cell receptor” or “engineered TCR” refers to aT-cell receptor (TCR) engineered to specifically bind with a desiredaffinity to a major histocompatibility complex (MHC)/peptide targetantigen that is selected, cloned, and/or subsequently introduced into apopulation of T-cells, often used for adoptive immunotherapy. Incontrast to engineered TCRs, CARs are engineered to bind target antigensin a MHC independent manner.

The term “expressed on” as used herein is used in reference to a proteinexpressed on the surface of a cell, such as a mammalian cell. Thus, theprotein is expressed as a membrane protein. In some embodiments, theexpressed protein is a transmembrane protein. In some embodiments, theprotein is conjugated to a small molecule moiety such as a drug ordetectable label. Proteins expressed on the surface of a cell caninclude cell-surface proteins such as cell surface receptors that areexpressed on mammalian cells.

The term “half-life extending moiety” refers to a moiety of apolypeptide fusion or chemical conjugate that extends the half-life of aprotein circulating in mammalian blood serum compared to the half-lifeof the protein that is not so conjugated to the moiety. In someembodiments, half-life is extended by greater than or greater than about1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold.In some embodiments, half-life is extended by more than 6 hours, morethan 12 hours, more than 24 hours, more than 48 hours, more than 72hours, more than 96 hours or more than 1 week after in vivoadministration compared to the protein without the half-life extendingmoiety. The half-life refers to the amount of time it takes for theprotein to lose half of its concentration, amount, or activity.Half-life can be determined for example, by using an ELISA assay or anactivity assay. Exemplary half-life extending moieties include an Fcdomain, a multimerization domain, polyethylene glycol (PEG),hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see,WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA),lipids (acylation), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid(glutamylation).

The term “immunological synapse” or “immune synapse” as used hereinmeans the interface between a mammalian cell that expresses MHC I (majorhistocompatibility complex) or MHC II, such as an antigen-presentingcell or tumor cell, and a mammalian lymphocyte such as an effector Tcell or Natural Killer (NK) cell.

An Fc (fragment crystallizable) region or domain of an immunoglobulinmolecule (also termed an Fc polypeptide) corresponds largely to theconstant region of the immunoglobulin heavy chain, and is responsiblefor various functions, including the antibody's effector function(s).The Fc domain contains part or all of a hinge domain of animmunoglobulin molecule plus a CH2 and a CH3 domain. The Fc domain canform a dimer of two polypeptide chains joined by one or more disulfidebonds. In some embodiments, the Fc is a variant Fc that exhibits reduced(e.g. reduced greater than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more)activity to facilitate an effector function. In some embodiments,reference to amino acid substitutions in an Fc region is by EU numberingsystem unless described with reference to a specific SEQ ID NO. EUnumbering is known and is according to the most recently updated IMGTScientific Chart (IMGT®, the international ImMunoGeneTics informationSystem®,http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html(created: 17 May 2001, last updated: 10 Jan. 2013) and the EU index asreported in Kabat, E. A. et al. Sequences of Proteins of Immunologicalinterest. 5th ed. US Department of Health and Human Services, NIHpublication No. 91-3242 (1991).

An immunoglobulin Fc fusion (“Fc-fusion”), such as an immunomodulatoryFc fusion protein, is a molecule comprising one or more polypeptides (orone or more small molecules) operably linked to an Fc region of animmunoglobulin. An Fc-fusion may comprise, for example, the Fc region ofan antibody (which facilitates pharmacokinetics) and a variant CD80polypeptide. An immunoglobulin Fc region may be linked indirectly ordirectly to one or more variant CD80 polypeptides or small molecules(fusion partners). Various linkers are known in the art and canoptionally be used to link an Fc to a fusion partner to generate anFc-fusion. Fc-fusions of identical species can be dimerized to formFc-fusion homodimers, or using non-identical species to form Fc-fusionheterodimers. In some embodiments, the Fc is a mammalian Fc such as amurine or human Fc.

The term “host cell” refers to a cell that can be used to express aprotein encoded by a recombinant expression vector. A host cell can be aprokaryote, for example, E. coli, or it can be a eukaryote, for example,a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media or CHO strain DX-B11,which is deficient in DHFR. In some embodiments, a host cell can be amammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a ratcell, a mouse cell, or an insect cell).

The term “immunoglobulin” (abbreviated “Ig”) as used herein refers to amammalian immunoglobulin protein including any of the five human classesof antibody: IgA (which includes subclasses IRA1 and IgA2), IgD, IgE,IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. Theterm is also inclusive of immunoglobulins that are less thanfull-length, whether wholly or partially synthetic (e.g., recombinant orchemical synthesis) or naturally produced, such as antigen bindingfragment (Fab), variable fragment (Fv) containing V_(H) and V_(L), thesingle chain variable fragment (scFv) containing V_(H) and V_(L) linkedtogether in one chain, as well as other antibody V region fragments,such as Fab′, F(ab)₂, F(ab′)₂, dsFv diabody, Fc, and Fd polypeptidefragments. Bispecific antibodies, homobispecific and heterobispecific,are included within the meaning of the term.

The term “immunoglobulin superfamily” or “IgSF” as used herein means thegroup of cell surface and soluble proteins that are involved in therecognition, binding, or adhesion processes of cells. Molecules arecategorized as members of this superfamily based on shared structuralfeatures with immunoglobulins (i.e., antibodies); they all possess adomain known as an immunoglobulin domain or fold. Members of the IgSFinclude cell surface antigen receptors, co-receptors and co-stimulatorymolecules of the immune system, molecules involved in antigenpresentation to lymphocytes, cell adhesion molecules, certain cytokinereceptors and intracellular muscle proteins. They are commonlyassociated with roles in the immune system. Proteins in theimmunological synapse are often members of the IgSF. IgSF can also beclassified into “subfamilies” based on shared properties such asfunction. Such subfamilies typically consist of from 4 to 30 IgSFmembers.

The terms “IgSF domain” or “immunoglobulin domain” or “Ig domain” asused herein refers to a structural domain of IgSF proteins. Ig domainsare named after the immunoglobulin molecules. They contain about 70-110amino acids and are categorized according to their size and function.Ig-domains possess a characteristic Ig-fold, which has a sandwich-likestructure formed by two sheets of antiparallel beta strands.Interactions between hydrophobic amino acids on the inner side of thesandwich and highly conserved disulfide bonds formed between cysteineresidues in the B and F strands, stabilize the Ig-fold. One end of theIg domain has a section called the complementarity determining regionthat is important for the specificity of antibodies for their ligands.The Ig like domains can be classified (into classes) as: IgV, IgC1,IgC2, or IgI. Most Ig domains are either variable (IgV) or constant(IgC). IgV domains with 9 beta strands are generally longer than IgCdomains with 7 beta strands. Ig domains of some members of the IgSFresemble IgV domains in the amino acid sequence, yet are similar in sizeto IgC domains. These are called IgC2 domains, while standard IgCdomains are called IgC1 domains. T-cell receptor (TCR) chains containtwo Ig domains in the extracellular portion; one IgV domain at theN-terminus and one IgC1 domain adjacent to the cell membrane. CD80contains two Ig domains: IgV and IgC.

The term “IgSF species” as used herein means an ensemble of IgSF memberproteins with identical or substantially identical primary amino acidsequence. Each mammalian immunoglobulin superfamily (IgSF) memberdefines a unique identity of all IgSF species that belong to that IgSFmember. Thus, each IgSF family member is unique from other IgSF familymembers and, accordingly, each species of a particular IgSF familymember is unique from the species of another IgSF family member.Nevertheless, variation between molecules that are of the same IgSFspecies may occur owing to differences in post-translationalmodification such as glycosylation, phosphorylation, ubiquitination,nitrosylation, methylation, acetylation, and lipidation. Additionally,minor sequence differences within a single IgSF species owing to genepolymorphisms constitute another form of variation within a single IgSFspecies as do wild type truncated forms of IgSF species owing to, forexample, proteolytic cleavage. A “cell surface IgSF species” is an IgSFspecies expressed on the surface of a cell, generally a mammalian cell.

The term “immunological activity” as used herein in the context ofmammalian lymphocytes such as T-cells refers to one or more cellsurvival, cell proliferation, cytokine production (e.g.interferon-gamma), or T-cell cytotoxicity activities. In some cases, animmunological activity can means their expression of cytokines, such aschemokines or interleukins. Assays for determining enhancement orsuppression of immunological activity include the MLR (mixed lymphocytereaction) assays measuring interferon-gamma cytokine levels in culturesupernatants (Wang et al., Cancer Immunol Res. 2014 Sep.: 2(9):846-56),SEB (staphylococcal enterotoxin B) T cell stimulation assay (Wang etal., Cancer Immunol Res. 2014 Sep.: 2(9):846-56), and anti-CD3 T cellstimulation assays (Li and Kurlander, J Transl Med. 2010: 8: 104). SinceT cell activation is associated with secretion of IFN-gamma cytokine,detecting IFN-gamma levels in culture supernatants from these in vitrohuman T cell assays can be assayed using commercial ELISA kits (Wu etal, Immunol Lett 2008 Apr. 15; 117(1): 57-62). Induction of an immuneresponse results in an increase in immunological activity relative toquiescent lymphocytes. An immunomodulatory protein, such as a variantCD80 polypeptide containing an affinity modified IgSF domain, asprovided herein can in some embodiments increase or, in alternativeembodiments, decrease IFN-gamma (interferon-gamma) expression in aprimary T-cell assay relative to a wild-type IgSF member or IgSF domaincontrol. Those of skill will recognize that the format of the primaryT-cell assay used to determine an increase in IFN-gamma expression willdiffer from that employed to assay for a decrease in IFN-gammaexpression. In assaying for the ability of an immunomodulatory proteinor affinity modified IgSF domain of the invention to decrease IFN-gammaexpression in a primary T-cell assay, a Mixed Lymphocyte Reaction (MLR)assay can be used as described in Example 6. Conveniently, a solubleform of an affinity modified IgSF domain of the invention can beemployed to determine its ability to antagonize and thereby decrease theIFN-gamma expression in a MLR as likewise described in Example 6.Alternatively, in assaying for the ability of an immunomodulatoryprotein or affinity modified IgSF domain of the invention to increaseIFN-gamma expression in a primary T-cell assay, a co-immobilizationassay can be used. In a co-immobilization assay, a T-cell receptorsignal, provided in some embodiments by anti-CD3 antibody, is used inconjunction with a co-immobilized affinity modified IgSF domain, such asa variant CD80, to determine the ability to increase IFN-gammaexpression relative to a wild-type IgSF domain control. Methods to assaythe immunological activity of engineered cells, including to evaluatethe activity of a variant CD80 transmembrane immunomodulatory protein,are known in the art and include, but are not limited to, the ability toexpand T cells following antigen stimulation, sustain T cell expansionin the absence of re-stimulation, and anti-cancer activities inappropriate animal models. Assays also include assays to assesscytotoxicity, including a standard ⁵¹Cr-release assay (see e.g. Miloneet al., (2009) Molecular Therapy 17: 1453-1464) or flow basedcytotoxicity assays, or an impedance based cytotoxicity assay (Peper etal. (2014) Journal of Immunological Methods, 405:192-198).

An “immunomodulatory polypeptide” is a polypeptide that modulatesimmunological activity. By “modulation” or “modulating” an immuneresponse is meant that immunological activity is either increased ordecreased. An immunomodulatory polypeptide can be a single polypeptidechain or a multimer (dimers or higher order multimers) of at least twopolypeptide chains covalently bonded to each other by, for example,interchain disulfide bonds. Thus, monomeric, dimeric, and higher ordermultimeric polypeptides are within the scope of the defined term.Multimeric polypeptides can be homomultimeric (of identical polypeptidechains) or heteromultimeric (of non-identical polypeptide chains). Animmunomodulatory polypeptide can comprise variant CD80.

The term “increase” as used herein means to increase by a statisticallysignificant amount. An increase can be at least 5%, 10%, 20%, 30%, 40%,50%, 75%, 100%, or greater than a non-zero control value.

An “isoform” of CD80 is one of a plurality of naturally occurring CD80polypeptides that differ in amino acid sequence. Isoforms can be theproduct of splice variants of an RNA transcript expressed by a singlegene, or the expression product of highly similar but different genesyielding a functionally similar protein such as may occur from geneduplication. As used herein, the term “isoform” of CD80 also refers tothe product of different alleles of a CD80 gene.

The term “lymphocyte” as used herein means any of three subtypes ofwhite blood cell in a mammalian immune system. They include naturalkiller cells (NK cells) (which function in cell-mediated, cytotoxicinnate immunity), T cells (for cell-mediated, cytotoxic adaptiveimmunity), and B cells (for humoral, antibody-driven adaptive immunity).T cells include: T helper cells, cytotoxic T-cells, natural killerT-cells, memory T-cells, regulatory T-cells, or gamma delta T-cells.Innate lymphoid cells (ILC) are also included within the definition oflymphocyte.

The terms “mammal,” or “patient” specifically includes reference to atleast one of a: human, chimpanzee, rhesus monkey, cynomolgus monkey,dog, cat, mouse, or rat.

The term “membrane protein” as used herein means a protein that, underphysiological conditions, is attached directly or indirectly to a lipidbilayer. A lipid bilayer that forms a membrane can be a biologicalmembrane such as a eukaryotic (e.g., mammalian) cell membrane or anartificial (i.e., man-made) membrane such as that found on a liposome.Attachment of a membrane protein to the lipid bilayer can be by way ofcovalent attachment, or by way of non-covalent interactions such ashydrophobic or electrostatic interactions. A membrane protein can be anintegral membrane protein or a peripheral membrane protein. Membraneproteins that are peripheral membrane proteins are non-covalentlyattached to the lipid bilayer or non-covalently attached to an integralmembrane protein. A peripheral membrane protein forms a temporaryattachment to the lipid bilayer such that under the range of conditionsthat are physiological in a mammal, peripheral membrane protein canassociate and/or disassociate from the lipid bilayer. In contrast toperipheral membrane proteins, integral membrane proteins form asubstantially permanent attachment to the membrane's lipid bilayer suchthat under the range of conditions that are physiological in a mammal,integral membrane proteins do not disassociate from their attachment tothe lipid bilayer. A membrane protein can form an attachment to themembrane by way of one layer of the lipid bilayer (monotopic), orattached by way of both layers of the membrane (polytopic). An integralmembrane protein that interacts with only one lipid bilayer is an“integral monotopic protein”. An integral membrane protein thatinteracts with both lipid bilayers is an “integral polytopic protein”alternatively referred to herein as a “transmembrane protein”.

The terms “modulating” or “modulate” as used herein in the context of animmune response, such as a mammalian immune response, refer to anyalteration, such as an increase or a decrease, of existing or potentialimmune responses that occurs as a result of administration of animmunomodulatory polypeptide comprising a variant CD80 of the presentinvention or as a result of administration of engineered cells expressesan immunomodulatory protein, such as a variant CD80 transmembraneimmunomodulatory protein of the present invention. Thus, it refers to analteration, such as an increase or decrease, of an immune response ascompared to the immune response that occurs or is present in the absenceof the administration of the immunomodulatory protein comprising thevariant CD80. Such modulation includes any induction, activation,suppression or alteration in degree or extent of immunological activityof an immune cell. Immune cells include B cells, T cells, NK (naturalkiller) cells, NK T cells, professional antigen-presenting cells (APCs),and non-professional antigen-presenting cells, and inflammatory cells(neutrophils, macrophages, monocytes, eosinophils, and basophils).Modulation includes any change imparted on an existing immune response,a developing immune response, a potential immune response, or thecapacity to induce, regulate, influence, or respond to an immuneresponse. Modulation includes any alteration in the expression and/orfunction of genes, proteins and/or other molecules in immune cells aspart of an immune response. Modulation of an immune response ormodulation of immunological activity includes, for example, thefollowing: elimination, deletion, or sequestration of immune cells;induction or generation of immune cells that can modulate the functionalcapacity of other cells such as autoreactive lymphocytes, antigenpresenting cells, or inflammatory cells; induction of an unresponsivestate in immune cells (i.e., anergy); enhancing or suppressing theactivity or function of immune cells, including but not limited toaltering the pattern of proteins expressed by these cells. Examplesinclude altered production and/or secretion of certain classes ofmolecules such as cytokines, chemokines, growth factors, transcriptionfactors, kinases, costimulatory molecules, or other cell surfacereceptors or any combination of these modulatory events. Modulation canbe assessed, for example, by an alteration in IFN-gamma (interferongamma) expression relative to the wild-type or unmodified CD80 controlin a primary T cell assay (see, Zhao and Ji, Exp Cell Res. 2016 Jan. 1;340(1) 132-138). Modulation can be assessed, for example, by analteration of an immunological activity of engineered cells, such as analteration in in cytotoxic activity of engineered cells or an alterationin cytokine secretion of engineered cells relative to cells engineeredwith a wild-type CD80 transmembrane protein.

The term, a “multimerization domain” refers to a sequence of amino acidsthat promotes stable interaction of a polypeptide molecule with one ormore additional polypeptide molecules, each containing a complementarymultimerization domain, which can be the same or a differentmultimerization domain to form a stable multimer with the first domain.Generally, a polypeptide is joined directly or indirectly to themultimerization domain. Exemplary multimerization domains include theimmunoglobulin sequences or portions thereof, leucine zippers,hydrophobic regions, hydrophilic regions, and compatible protein-proteininteraction domains. The multimerization domain, for example, can be animmunoglobulin constant region or domain, such as, for example, the Fcdomain or portions thereof from IgG, including IgG1, IgG2, IgG3 or IgG4subtypes, IgA, IgE, IgD and IgM and modified forms thereof.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyto refer to a polymer of nucleic acid residues (e.g.,deoxyribonucleotides or ribonucleotides) in either single- ordouble-stranded form. Unless specifically limited, the terms encompassnucleic acids containing known analogues of natural nucleotides and thathave similar binding properties to it and are metabolized in a mannersimilar to naturally-occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary nucleotide sequences as well as thesequence explicitly indicated (a “reference sequence”). Specifically,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues. The termnucleic acid or polynucleotide encompasses cDNA or mRNA encoded by agene.

The term “molecular species” as used herein means an ensemble ofproteins with identical or substantially identical primary amino acidsequence. Each mammalian immunoglobulin superfamily (IgSF) memberdefines a collection of identical or substantially identical molecularspecies. Thus, for example, human CD80 is an IgSF member and each humanCD80 molecule is a molecular species of CD80. Variation betweenmolecules that are of the same molecular species may occur owing todifferences in post-translational modification such as glycosylation,phosphorylation, ubiquitination, nitrosylation, methylation,acetylation, and lipidation. Additionally, minor sequence differenceswithin a single molecular species owing to gene polymorphisms constituteanother form of variation within a single molecular species as do wildtype truncated forms of a single molecular species owing to, forexample, proteolytic cleavage. A “cell surface molecular species” is amolecular species expressed on the surface of a mammalian cell. Two ormore different species of protein, each of which is present exclusivelyon one or exclusively the other (but not both) of the two mammaliancells forming the IS, are said to be in “cis” or “cis configuration”with each other. Two different species of protein, the first of which isexclusively present on one of the two mammalian cells forming the IS andthe second of which is present exclusively on the second of the twomammalian cells forming the IS, are said to be in “trans” or “transconfiguration.” Two different species of protein each of which ispresent on both of the two mammalian cells forming the IS are in bothcis and trans configurations on these cells.

The term “non-competitive binding” as used herein means the ability of aprotein to specifically bind simultaneously to at least two cognatebinding partners. Thus, the protein is able to bind to at least twodifferent cognate binding partners at the same time, although thebinding interaction need not be for the same duration such that, in somecases, the protein is specifically bound to only one of the cognatebinding partners. In some embodiments, the binding occurs under specificbinding conditions. In some embodiments, the simultaneous binding issuch that binding of one cognate binding partner does not substantiallyinhibit simultaneous binding to a second cognate binding partner. Insome embodiments, non-competitive binding means that binding a secondcognate binding partner to its binding site on the protein does notdisplace the binding of a first cognate binding partner to its bindingsite on the protein. Methods of assessing non-competitive binding arewell known in the art such as the method described in Perez de La Lastraet al., Immunology, 1999 April: 96(4): 663-670. In some cases, innon-competitive interactions, the first cognate binding partnerspecifically binds at an interaction site that does not overlap with theinteraction site of the second cognate binding partner such that bindingof the second cognate binding partner does not directly interfere withthe binding of the first cognate binding partner. Thus, any effect onbinding of the cognate binding partner by the binding of the secondcognate binding partner is through a mechanism other than directinterference with the binding of the first cognate binding partner. Forexample, in the context of enzyme-substrate interactions, anon-competitive inhibitor binds to a site other than the active site ofthe enzyme. Non-competitive binding encompasses uncompetitive bindinginteractions in which a second cognate binding partner specificallybinds at an interaction site that does not overlap with the binding ofthe first cognate binding partner but binds to the second interactionsite only when the first interaction site is occupied by the firstcognate binding partner.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a mammalian subject, often a human. Apharmaceutical composition typically comprises an effective amount of anactive agent (e.g., an immunomodulatory polypeptide comprising a variantCD80 or engineered cells expressing a variant CD80 transmembraneimmunomodulatory protein) and a carrier, excipient, or diluent. Thecarrier, excipient, or diluent is typically a pharmaceuticallyacceptable carrier, excipient or diluent, respectively.

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to a molecular chain of two or more amino acids linked throughpeptide bonds. The terms do not refer to a specific length of theproduct. Thus, “peptides,” and “oligopeptides,” are included within thedefinition of polypeptide. The terms include post-translationalmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. The terms also includemolecules in which one or more amino acid analogs or non-canonical orunnatural amino acids that can be synthesized, or expressedrecombinantly using known protein engineering techniques. In addition,proteins can be derivatized.

The term “primary T-cell assay” as used herein refers to an in vitroassay to measure interferon-gamma (“IFN-gamma”) expression. A variety ofsuch primary T-cell assays are known in the art such as that describedin Example 6. In a preferred embodiment, the assay used is anti-CD3coimmobilization assay. In this assay, primary T cells are stimulated byanti-CD3 immobilized with or without additional recombinant proteins.Culture supernatants are harvested at timepoints, usually 24-72 hours.In another embodiment, the assay used is a mixed lymphocyte reaction(MLR). In this assay, primary T cells are simulated with allogenic APC.Culture supernatants are harvested at timepoints, usually 24-72 hours.Human IFN-gamma levels are measured in culture supernatants by standardELISA techniques. Commercial kits are available from vendors and theassay is performed according to manufacturer's recommendation.

The term “purified” as applied to nucleic acids, such as encodingimmunomodulatory proteins of the invention, generally denotes a nucleicacid or polypeptide that is substantially free from other components asdetermined by analytical techniques well known in the art (e.g., apurified polypeptide or polynucleotide forms a discrete band in anelectrophoretic gel, chromatographic eluate, and/or a media subjected todensity gradient centrifugation). For example, a nucleic acid orpolypeptide that gives rise to essentially one band in anelectrophoretic gel is “purified.” A purified nucleic acid or protein ofthe invention is at least about 50% pure, usually at least about 75%,80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., percent by weight or ona molar basis).

The term “recombinant” indicates that the material (e.g., a nucleic acidor a polypeptide) has been artificially (i.e., non-naturally) altered byhuman intervention. The alteration can be performed on the materialwithin, or removed from, its natural environment or state. For example,a “recombinant nucleic acid” is one that is made by recombining nucleicacids, e.g., during cloning, affinity modification, DNA shuffling orother well-known molecular biological procedures. A “recombinant DNAmolecule,” is comprised of segments of DNA joined together by means ofsuch molecular biological techniques. The term “recombinant protein” or“recombinant polypeptide” as used herein refers to a protein moleculewhich is expressed using a recombinant DNA molecule. A “recombinant hostcell” is a cell that contains and/or expresses a recombinant nucleicacid or that is otherwise altered by genetic engineering, such as byintroducing into the cell a nucleic acid molecule encoding a recombinantprotein, such as a transmembrane immunomodulatory protein providedherein. Transcriptional control signals in eukaryotes comprise“promoter” and “enhancer” elements. Promoters and enhancers consist ofshort arrays of DNA sequences that interact specifically with cellularproteins involved in transcription. Promoter and enhancer elements havebeen isolated from a variety of eukaryotic sources including genes inyeast, insect and mammalian cells and viruses (analogous controlelements, i.e., promoters, are also found in prokaryotes). The selectionof a particular promoter and enhancer depends on what cell type is to beused to express the protein of interest. The terms “in operablecombination,” “in operable order” and “operably linked” as used hereinrefer to the linkage of nucleic acid sequences in such a manner ororientation that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced.

The term “recombinant expression vector” as used herein refers to a DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host cell. Nucleic acid sequencesnecessary for expression in prokaryotes include a promoter, optionallyan operator sequence, a ribosome binding site and possibly othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals. A secretory signal peptidesequence can also, optionally, be encoded by the recombinant expressionvector, operably linked to the coding sequence for the recombinantprotein, such as a recombinant fusion protein, so that the expressedfusion protein can be secreted by the recombinant host cell, for easierisolation of the fusion protein from the cell, if desired. The termincludes the vector as a self-replicating nucleic acid structure as wellas the vector incorporated into the genome of a host cell into which ithas been introduced. Among the vectors are viral vectors, such aslentiviral vectors.

The term “selectivity” refers to the preference of a subject protein, orpolypeptide, for specific binding of one substrate, such as one cognatebinding partner, compared to specific binding for another substrate,such as a different cognate binding partner of the subject protein.Selectivity can be reflected as a ratio of the binding activity (e.g.binding affinity) of a subject protein and a first substrate, such as afirst cognate binding partner, (e.g., Kai) and the binding activity(e.g. binding affinity) of the same subject protein with a secondcognate binding partner (e.g., K_(d2)).

The term “sequence identity” as used herein refers to the sequenceidentity between genes or proteins at the nucleotide or amino acidlevel, respectively. “Sequence identity” is a measure of identitybetween proteins at the amino acid level and a measure of identitybetween nucleic acids at nucleotide level. The protein sequence identitymay be determined by comparing the amino acid sequence in a givenposition in each sequence when the sequences are aligned. Similarly, thenucleic acid sequence identity may be determined by comparing thenucleotide sequence in a given position in each sequence when thesequences are aligned. Methods for the alignment of sequences forcomparison are well known in the art, such methods include GAP, BESTFIT,BLAST, FASTA and TFASTA, The BLAST algorithm calculates percent sequenceidentity and performs a statistical analysis of the similarity betweenthe two sequences. The software for performing BLAST analysis ispublicly available through the National Center for BiotechnologyInformation (NCBI) website.

The term “soluble” as used herein in reference to proteins, means thatthe protein is not a membrane protein. In general, a soluble proteincontains only the extracellular domain of an IgSF family memberreceptor, or a portion thereof containing an IgSF domain or domains orspecific-binding fragments thereof, but does not contain thetransmembrane domain. In some cases, solubility of a protein can beimproved by linkage or attachment, directly or indirectly via a linker,to an Fc domain, which, in some cases, also can improve the stabilityand/or half-life of the protein. In some aspects, a soluble protein isan Fc fusion protein.

The term “species” as used herein with respect to polypeptides ornucleic acids means an ensemble of molecules with identical orsubstantially identical sequences. Variation between polypeptides thatare of the same species may occur owing to differences inpost-translational modification such as glycosylation, phosphorylation,ubiquitination, nitrosylation, methylation, acetylation, and lipidation.Slightly truncated sequences of polypeptides that differ (or encode adifference) from the full length species at the amino-terminus orcarboxyl-terminus by no more than 1, 2, or 3 amino acid residues areconsidered to be of a single species. Such microheterogeneities are acommon feature of manufactured proteins.

The term “specific binding fragment” as used herein in reference to afull-length wild-type mammalian CD80 polypeptide or an IgV or an IgCdomain thereof, means a polypeptide having a subsequence of an IgVand/or IgC domain and that specifically binds in vitro and/or in vivo toa mammalian CD28, mammalian PD-L1 and/or mammalian CTLA-4, such as ahuman or murine CD28, PD-L1, and/or CTLA-4. In some embodiments, thespecific binding fragment of the CD80 IgV or the CD80 IgC is at least60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% the sequencelength of the full-length wild-type sequence. The specific bindingfragment can be altered in sequence to form the variant CD80.

The term “specifically binds” as used herein means the ability of aprotein, under specific binding conditions, to bind to a target proteinsuch that its affinity or avidity is at least 5 times as great, butoptionally at least 10, 20, 30, 40, 50, 100, 250 or 500 times as great,or even at least 1000 times as great as the average affinity or avidityof the same protein to a collection of random peptides or polypeptidesof sufficient statistical size. A specifically binding protein need notbind exclusively to a single target molecule but may specifically bindto a non-target molecule due to similarity in structural conformationbetween the target and non-target (e.g., paralogs or orthologs). Thoseof skill will recognize that specific binding to a molecule having thesame function in a different species of animal (i.e., ortholog) or to anon-target molecule having a substantially similar epitope as the targetmolecule (e.g., paralog) is possible and does not detract from thespecificity of binding which is determined relative to a statisticallyvalid collection of unique non-targets (e.g., random polypeptides).Thus, a polypeptide of the invention may specifically bind to more thanone distinct species of target molecule due to cross-reactivity.Solid-phase ELISA immunoassays or Biacore measurements can be used todetermine specific binding between two proteins. Generally, interactionsbetween two binding proteins have dissociation constants (K_(d)) lessthan 1×10⁻⁵M, and often as low as 1×10⁻¹² M. In certain embodiments ofthe present disclosure, interactions between two binding proteins havedissociation constants of 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M,1×10⁻¹⁰ M or 1×10⁻¹¹ M.

The terms “surface expresses” or “surface expression” in reference to amammalian cell expressing a polypeptide means that the polypeptide isexpressed as a membrane protein. In some embodiments, the membraneprotein is a transmembrane protein.

As used herein, “synthetic,” with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

The term “targeting moiety” as used herein refers to a composition thatis covalently or non-covalently attached to, or physically encapsulates,a polypeptide comprising the variant CD80. The targeting moiety hasspecific binding affinity for a desired counter-structure such as a cellsurface receptor (e.g., the B7 family member PD-L1), or a tumor antigensuch as tumor specific antigen (TSA) or a tumor associated antigen (TAA)such as B7-H6. Typically, the desired counter-structure is localized ona specific tissue or cell-type. Targeting moieties include: antibodies,antigen binding fragment (Fab), variable fragment (Fv) containing V_(H)and V_(L), the single chain variable fragment (scFv) containing V_(H)and V_(L) linked together in one chain, as well as other antibody Vregion fragments, such as Fab′, F(ab)₂, F(ab′)₂, dsFv diabody,nanobodies, soluble receptors, receptor ligands, affinity maturedreceptors or ligands, as well as small molecule (<500 dalton)compositions (e.g., specific binding receptor compositions). Targetingmoieties can also be attached covalently or non-covalently to the lipidmembrane of liposomes that encapsulate a polypeptide of the presentinvention.

The term “transmembrane protein” as used herein means a membrane proteinthat substantially or completely spans a lipid bilayer such as thoselipid bilayers found in a biological membrane such as a mammalian cell,or in an artificial construct such as a liposome. The transmembraneprotein comprises a transmembrane domain (“transmembrane domain”) bywhich it is integrated into the lipid bilayer and by which theintegration is thermodynamically stable under physiological conditions.Transmembrane domains are generally predictable from their amino acidsequence via any number of commercially available bioinformaticssoftware applications on the basis of their elevated hydrophobicityrelative to regions of the protein that interact with aqueousenvironments (e.g., cytosol, extracellular fluid). A transmembranedomain is often a hydrophobic alpha helix that spans the membrane. Atransmembrane protein can pass through the both layers of the lipidbilayer once or multiple times. A transmembrane protein includes theprovided transmembrane immunomodulatory proteins described herein. Inaddition to the transmembrane domain, a transmembrane immunomodulatoryprotein of the invention further comprises an ectodomain and, in someembodiments, an endodomain.

The terms “treating,” “treatment,” or “therapy” of a disease or disorderas used herein mean slowing, stopping or reversing the disease ordisorders progression, as evidenced by decreasing, cessation orelimination of either clinical or diagnostic symptoms, by administrationof a therapeutic composition (e.g. containing an immunomodulatoryprotein or engineered cells) of the invention either alone or incombination with another compound as described herein. “Treating,”“treatment,” or “therapy” also means a decrease in the severity ofsymptoms in an acute or chronic disease or disorder or a decrease in therelapse rate as for example in the case of a relapsing or remittingautoimmune disease course or a decrease in inflammation in the case ofan inflammatory aspect of an autoimmune disease. As used herein in thecontext of cancer, the terms “treatment” or, “inhibit,” “inhibiting” or“inhibition” of cancer refers to at least one of: a statisticallysignificant decrease in the rate of tumor growth, a cessation of tumorgrowth, or a reduction in the size, mass, metabolic activity, or volumeof the tumor, as measured by standard criteria such as, but not limitedto, the Response Evaluation Criteria for Solid Tumors (RECIST), or astatistically significant increase in progression free survival (PFS) oroverall survival (OS). “Preventing,” “prophylaxis,” or “prevention” of adisease or disorder as used in the context of this invention refers tothe administration of an immunomodulatory polypeptide or engineeredcells of the invention, either alone or in combination with anothercompound, to prevent the occurrence or onset of a disease or disorder orsome or all of the symptoms of a disease or disorder or to lessen thelikelihood of the onset of a disease or disorder.

The term “tumor specific antigen” or “TSA” as used herein refers to acounter-structure that is present primarily on tumor cells of amammalian subject but generally not found on normal cells of themammalian subject. A tumor specific antigen need not be exclusive totumor cells but the percentage of cells of a particular mammal that havethe tumor specific antigen is sufficiently high or the levels of thetumor specific antigen on the surface of the tumor are sufficiently highsuch that it can be targeted by anti-tumor therapeutics, such asimmunomodulatory polypeptides of the invention, and provide preventionor treatment of the mammal from the effects of the tumor. In someembodiments, in a random statistical sample of cells from a mammal witha tumor, at least 50% of the cells displaying a TSA are cancerous. Inother embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of thecells displaying a TSA are cancerous.

The term “variant” (also “modified” or mutant”) as used in reference toa variant CD80 means a CD80, such as a mammalian (e.g., human or murine)CD80 created by human intervention. The variant CD80 is a polypeptidehaving an altered amino acid sequence, relative to an unmodified orwild-type CD80. The variant CD80 is a polypeptide which differs from awild-type CD80 isoform sequence by one or more amino acid substitutions,deletions, additions, or combinations thereof. For purposes herein, thevariant CD80 contains at least one affinity modified domain, whereby oneor more of the amino acid differences occurs in an IgSF domain (e.g. IgVdomain). A variant CD80 can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30 or more amino acid differences, such as amino acid substitutions. Avariant CD80 polypeptide generally exhibits at least 50%, 60%, 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to a corresponding wild-type or unmodifiedCD80, such as to the sequence of SEQ ID NO:1, a mature sequence thereofor a portion thereof containing the extracellular domain or an IgSFdomain thereof. In some embodiments, a variant CD80 polypeptide exhibitsat least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to acorresponding wild-type or unmodified CD80 comprising the sequence setforth in SEQ ID NO:28, SEQ ID NO: 152, or SEQ ID NO:372. Non-naturallyoccurring amino acids as well as naturally occurring amino acids areincluded within the scope of permissible substitutions or additions. Avariant CD80 is not limited to any particular method of making andincludes, for example, de novo chemical synthesis, de novo recombinantDNA techniques, or combinations thereof. A variant CD80 of the inventionspecifically binds to at least one or more of: CD28, PD-L1 and/or CTLA-4of a mammalian species. In some embodiments, the altered amino acidsequence results in an an altered (i.e., increased or decreased) bindingaffinity or avidity to CD28, PD-L1 and/or CTLA-4 compared to theunmodified or wild-type CD80 protein. An increase or decrease in bindingaffinity or avidity can be determined using well known binding assayssuch as flow cytometry. Larsen et al., American Journal ofTransplantation, Vol 5: 443-453 (2005). See also, Linsley et al.,Immunity, 1: 7930801 (1994). An increase in variant CD80 bindingaffinity or avidity to CD28, PD-L1 and/or CTLA-4 can be a value at least5% greater than that of the unmodified or wild-type CD80 and in someembodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater thanthat of the unmodified or wild-type CD80 control value. A decrease inCD80 binding affinity or avidity to CD28, PD-L1 and/or CTLA-4 is to avalue no greater than 95% of the of the unmodified or wild-type CD80control values, and in some embodiments no greater than 80%, 70% 60%,50%, 40%, 30%, 20%, 10%, 5%, or no detectable binding affinity oravidity of the unmodified or wild-type CD80 control values. A variantCD80 polypeptide is altered in primary amino acid sequence bysubstitution, addition, or deletion of amino acid residues. The term“variant” in the context of variant CD80 polypeptide is not be construedas imposing any condition for any particular starting composition ormethod by which the variant CD80 is created. A variant CD80 can, forexample, be generated starting from wild type mammalian CD80 sequenceinformation, then modeled in silico for binding to CD28, PD-L1 and/orCTLA-4, and finally recombinantly or chemically synthesized to yield thevariant CD80. In but one alternative example, the variant CD80 can becreated by site-directed mutagenesis of an unmodified or wild-type CD80.Thus, variant CD80 denotes a composition and not necessarily a productproduced by any given process. A variety of techniques includingrecombinant methods, chemical synthesis, or combinations thereof, may beemployed.

The term “wild-type” or “natural” or “native” as used herein is used inconnection with biological materials such as nucleic acid molecules,proteins (e.g., CD80), IgSF members, host cells, and the like, refers tothose which are found in nature and not modified by human intervention.

II. VARIANT CD80 POLYPEPTIDES

Provided herein are variant CD80 polypeptides that exhibit altered(increased or decreased) binding activity or affinity for one or moreCD80 cognate binding partners. In some embodiments, the CD80 cognatebinding partner is CD28, PD-L1, or CTLA-4. In some embodiments, thevariant CD80 polypeptide contains one or more amino acid modifications,such as one or more substitutions (alternatively, “mutations” or“replacements”), deletions or additions in an immunoglobulin superfamily(IgSF) domain (IgD) relative to a wild-type or unmodified CD80polypeptide or a portion of a wild-type or unmodified CD80 containingthe IgD or a specific binding fragment thereof.

Thus, a provided variant CD80 polypeptide is or comprises a variant IgD(hereinafter called “vIgD”) in which the one or more amino acidmodifications (e.g., substitutions) is in an IgD.

In some embodiments, the IgD comprises an IgV domain or an IgC (e.g.IgC2) domain or specific binding fragment of the IgV domain or the IgC(e.g. IgC2) domain, or combinations thereof. In some embodiments, theIgD can be an IgV only, the combination of the IgV and IgC, includingthe entire extracellular domain (ECD), or any combination of Ig domainsof CD80. Table 2 provides exemplary residues that correspond to IgV orIgC regions of CD80. In some embodiments, the variant CD80 polypeptidecontains an IgV domain, or an IgC domain, or specific binding fragmentsthereof in which the at least one amino acid modification (e.g.,substitution) in the IgV domain or IgC domain or the specific bindingfragment thereof. In some embodiments, the variant CD80 polypeptidecontains an IgV domain or specific binding fragments thereof in whichthe at least one of the amino acid modifications (e.g., substitutions)is in the IgV domain or a specific binding fragment thereof. In someembodiments, by virtue of the altered binding activity or affinity, thealtered IgV domain or IgC domain is an affinity modified IgSF domain.

In some embodiments, the variant is modified in one more IgSF domainsrelative to the sequence of an unmodified CD80 sequence. In someembodiments, the unmodified CD80 sequence is a wild-type CD80. In someembodiments, the unmodified or wild-type CD80 has the sequence of anative CD80 or an ortholog thereof. In some embodiments, the unmodifiedCD80 is or comprises the extracellular domain (ECD) of CD80 or a portionthereof containing one or more IgSF domain (see Table 2). In someembodiments, the extracellular domain of an unmodified or wild-type CD80polypeptide comprises an IgV domain and an IgC domain or domains.However, the variant CD80 polypeptide need not comprise both the IgVdomain and the IgC domain or domains. In some embodiments, the variantCD80 polypeptide comprises or consists essentially of the IgV domain ora specific binding fragment thereof. In some embodiments, the variantCD80 polypeptide comprises or consists essentially of the IgC domain orspecific binding fragments thereof. In some embodiments, the variantCD80 is soluble and lacks a transmembrane domain. In some embodiments,the variant CD80 further comprises a transmembrane domain and, in somecases, also a cytoplasmic domain.

In some embodiments, the wild-type or unmodified CD80 polypeptide is amammalian CD80 polypeptide, such as, but not limited to, a human, amouse, a cynomolgus monkey, or a rat CD80 polypeptide. In someembodiments, the wild-type or unmodified CD80 sequence is human.

In some embodiments, the wild-type or unmodified CD80 polypeptide has(i) the sequence of amino acids set forth in SEQ ID NO: 1 or a matureform thereof lacking the signal sequence, (ii) a sequence of amino acidsthat exhibits at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1 or amature form thereof, or (iii) is a portion of (i) or (ii) containing anIgV domain or IgC domain or specific binding fragments thereof.

In some embodiments, the wild-type or unmodified CD80 polypeptide is orcomprises an extracellular domain of the CD80 or a portion thereof. Forexample, in some embodiments, the unmodified or wild-type CD80polypeptide comprises the amino acid sequence set forth in SEQ ID NO:28, or an ortholog thereof. For example, the unmodified or wild-typeCD80 polypeptide can comprise (i) the sequence of amino acids set forthin SEQ ID NO:28, (ii) a sequence of amino acids that has at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% sequence identity to SEQ ID NO: 28, or (iii) is a specific bindingfragment of of (i) or (ii) comprising an IgV domain or an IgC domain. Insome embodiments, the wild-type or unmodified extracellular domain ofCD80 is capable of binding one or more CD80 cognate binding proteins,such as one or more of CD28, PD-L1 or CTLA-4.

In some embodiments, the wild-type or unmodified CD80 polypeptidecontains an IgV domain or an IgC domain, or a specific binding fragmentthereof. In some embodiments, the IgV domain of the wild-type orunmodified CD80 polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 152 or 372, or an ortholog thereof. For example, the IgVdomain of the unmodified or wild-type CD80 polypeptide can contain (i)the sequence of amino acids set forth in SEQ ID NO: 152 or 372, (ii) asequence of amino acids that has at least about 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity toSEQ ID NO: 152 or 372, or (iii) is a specific binding fragment of (i) or(ii). In some embodiments, the wild-type or unmodified IgV domain iscapable of binding one or more CD80 cognate binding proteins, such asone or more of CD28, PD-L1 or CTLA-4.

In some embodiments, the IgC domain of the wild-type or unmodified CD80polypeptide comprises the amino acid sequence set forth as residues145-230 or 154-232 of SEQ ID NO: 1, or an ortholog thereof. For example,the IgC domain of the unmodified or wild-type CD80 polypeptide cancontain (i) the sequence of amino acids set forth residues 145-230 or154-232 of SEQ ID NO: 1, (ii) a sequence of amino acids that has atleast about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% sequence identity to residues 145-230 or 154-232 of SEQ IDNO: 1, or (iii) is a specific binding fragment of (i) or (ii). In someembodiments, the wild-type or unmodified IgC domain is capable ofbinding one or more CD80 cognate binding proteins.

In some embodiments, the wild-type or unmodified CD80 polypeptidecontains a specific binding fragment of CD80, such as a specific bindingfragment of the IgV domain or the IgC domain. In some embodiments thespecific binding fragment can bind CD28, PD-L1, and/or CTLA-4. Thespecific binding fragment can have an amino acid length of at least 50amino acids, such as at least 60, 70, 80, 90, 100, or 110 amino acids.In some embodiments, the specific binding fragment of the IgV domaincontains an amino acid sequence that is at least about 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the lengthof the IgV domain set forth as amino acids 35-135 or 37-138 of SEQ IDNO: 1. In some embodiments, the specific binding fragment of the IgCdomain comprises an amino acid sequence that is at least about 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of thelength of the IgC domain set forth as amino acids 145-230 or 154-232 ofSEQ ID NO: 1.

In some embodiments, the variant CD80 polypeptide comprises the ECDdomain or a portion thereof comprising one or more affinity modifiedIgSF domains. In some embodiments, the variant CD80 polypeptides cancomprise an IgV domain or an IgC domain, or a specific binding fragmentof the IgV domain or a specific binding fragment of the IgC domain inwhich at least one of the IgV or IgC domain contains the one or moreamino acid modifications (e.g., substitutions). In some embodiments, thevariant CD80 polypeptides can comprise an IgV domain and an IgC domain,or a specific binding fragment of the IgV domain and a specific bindingfragment of the IgC domain. In some embodiments, the variant CD80polypeptide comprises a full-length IgV domain. In some embodiments, thevariant CD80 polypeptide comprises a full-length IgC domain. In someembodiments, the variant CD80 polypeptide comprises a specific bindingfragment of the IgV domain. In some embodiments, the variant CD80polypeptide comprises a specific binding fragment of the IgC domain. Insome embodiments, the variant CD80 polypeptide comprises a full-lengthIgV domain and a full-length IgC domain. In some embodiments, thevariant CD80 polypeptide comprises a full-length IgV domain and aspecific binding fragment of an IgC domain. In some embodiments, thevariant CD80 polypeptide comprises a specific binding fragment of an IgVdomain and a full-length IgC domain. In some embodiments, the variantCD80 polypeptide comprises a specific binding fragment of an IgV domainand a specific binding fragment of an IgC domain.

In any of such embodiments, the one or more amino acid modifications(e.g., substitutions) of the variant CD80 polypeptides can be located inany one or more of the CD80 polypeptide domains. For example, in someembodiments, one or more amino acid modifications (e.g., substitutions)are located in the extracellular domain of the variant CD80 polypeptide.In some embodiments, one or more amino acid modifications (e.g.,substitutions) are located in the IgV domain or specific bindingfragment of the IgV domain. In some embodiments, one or more amino acidmodifications (e.g., substitutions) are located in the IgC domain orspecific binding fragment of the IgC domain.

Generally, each of the various attributes of polypeptides are separatelydisclosed below (e.g., soluble and membrane bound polypeptides, affinityof CD80 for CD28, PD-L1, and CTLA-4, number of variations perpolypeptide chain, number of linked polypeptide chains, the number andnature of amino acid alterations per variant CD80, etc.). However, aswill be clear to the skilled artisan, any particular polypeptide cancomprise a combination of these independent attributes. It is understoodthat reference to amino acids, including to a specific sequence setforth as a SEQ ID NO used to describe domain organization of an IgSFdomain are for illustrative purposes and are not meant to limit thescope of the embodiments provided. It is understood that polypeptidesand the description of domains thereof are theoretically derived basedon homology analysis and alignments with similar molecules. Thus, theexact locus can vary, and is not necessarily the same for each protein.Hence, the specific IgSF domain, such as specific IgV domain or IgCdomain, can be several amino acids (such as one, two, three or four)longer or shorter.

Further, various embodiments of the invention as discussed below arefrequently provided within the meaning of a defined term as disclosedabove. The embodiments described in a particular definition aretherefore to be interpreted as being incorporated by reference when thedefined term is utilized in discussing the various aspects andattributes described herein. Thus, the headings, the order ofpresentation of the various aspects and embodiments, and the separatedisclosure of each independent attribute is not meant to be a limitationto the scope of the present disclosure.

EXEMPLARY MODIFICATIONS

Provided herein are variant CD80 polypeptides containing at least oneaffinity-modified IgSF domain (e.g. IgV or IgC) or a specific bindingfragment thereof relative to an IgSF domain contained in a wild-type orunmodified CD80 polypeptide such that the variant CD80 polypeptideexhibits altered (increased or decreased) binding activity or affinityfor one or more ligands CD28, PD-L1 or CTLA-4 compared to a wild-type orunmodified CD80 polypeptide. In some embodiments, a variant CD80polypeptide has a binding affinity for CD28, PD-L1, and/or CTLA-4 thatdiffers from that of a wild-type or unmodified CD80 polypeptide controlsequence as determined by, for example, solid-phase ELISA immunoassays,flow cytometry or Biacore assays. In some embodiments, the variant CD80polypeptide has an increased binding affinity for CD28, PD-L1, and/orCTLA-4. In some embodiments, the variant CD80 polypeptide has adecreased binding affinity for CD28, PD-L1, and/or CTLA-4, relative to awild-type or unmodified CD80 polypeptide. The CD28, PD-L1 and/or theCTLA-4 can be a mammalian protein, such as a human protein or a murineprotein.

Binding affinities for each of the cognate binding partners areindependent; that is, in some embodiments, a variant CD80 polypeptidehas an increased binding affinity for one, two or three of CD28, PD-L1,and CTLA-4, and/or a decreased binding affinity for one, two or three ofCD28, PD-L1, and CTLA-4, relative to a wild-type or unmodified CD80polypeptide.

In some embodiments, the variant CD80 polypeptide has an increasedbinding affinity for CD28, relative to a wild-type or unmodified CD80polypeptide. In some embodiments, the variant CD80 polypeptide has anincreased binding affinity for PD-L1, relative to a wild-type orunmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has an increased binding affinity for CTLA-4, relative to awild-type or unmodified CD80 polypeptide. In some embodiments, thevariant CD80 polypeptide has a decreased binding affinity for CD28,relative to a wild-type or unmodified CD80 polypeptide. In someembodiments, the variant CD80 polypeptide has a decreased bindingaffinity for PD-L1, relative to a wild-type or unmodified CD80polypeptide. In some embodiments, the variant CD80 polypeptide has adecreased binding affinity for CTLA-4, relative to a wild-type orunmodified CD80 polypeptide.

In some embodiments, the variant CD80 polypeptide has an increasedbinding affinity for CD28 and PD-L1, relative to a wild-type orunmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has an increased binding affinity for CD28 and a decreasedbinding affinity for PD-L1, relative to a wild-type or unmodified CD80polypeptide. In some embodiments, the variant CD80 polypeptide has adecreased binding affinity for CD28 and PD-L1, relative to a wild-typeor unmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has a decreased binding affinity for CD28 and an increasedbinding affinity for PD-L1, relative to a wild-type or unmodified CD80polypeptide.

In some embodiments, the variant CD80 polypeptide has an increasedbinding affinity for CD28 and CTLA-4, relative to a wild-type orunmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has an increased binding affinity for CD28 and a decreasedbinding affinity for CTLA-4, relative to a wild-type or unmodified CD80polypeptide. In some embodiments, the variant CD80 polypeptide has adecreased binding affinity for CD28 and CTLA-4, relative to a wild-typeor unmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has a decreased binding affinity for CD28 and an increasedbinding affinity for CTLA-4, relative to a wild-type or unmodified CD80polypeptide.

In some embodiments, the variant CD80 polypeptide has an increasedbinding affinity for PD-L1 and CTLA-4, relative to a wild-type orunmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has an increased binding affinity for PD-L1 and a decreasedbinding affinity for CTLA-4, relative to a wild-type or unmodified CD80polypeptide. In some embodiments, the variant CD80 polypeptide has adecreased binding affinity for PD-L1 and CTLA-4, relative to a wild-typeor unmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has a decreased binding affinity for PD-L1 and an increasedbinding affinity for CTLA-4, relative to a wild-type or unmodified CD80polypeptide.

In some embodiments, the variant CD80 polypeptide has an increasedbinding affinity for CD28, PD-L1, and CTLA-4, relative to a wild-type orunmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has an increased binding affinity for CD28 and PD-L1, and adecreased binding affinity for CTLA-4, relative to a wild-type orunmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has an increased binding affinity for CD28 and CTLA-4, and adecreased binding affinity for PD-L1, relative to a wild-type orunmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has a decreased binding affinity for CD28 and PD-L1, and anincreased binding affinity for CTLA-4, relative to a wild-type orunmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has a decreased binding affinity for CD28 and an increasedbinding affinity for PD-L1 and CTLA-4, relative to a wild-type orunmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has an increased binding affinity for CD28, and a decreasedbinding affinity for PD-L1 and CTLA-4, relative to a wild-type orunmodified CD80 polypeptide. In some embodiments, the variant CD80polypeptide has a decreased binding affinity for CD28, CTLA-4, andPD-L1, relative to a wild-type or unmodified CD80 polypeptide. In someembodiments, the variant CD80 polypeptide has a decreased bindingaffinity for CD28, and an increased binding affinity for PD-L1 andCTLA-4, relative to a wild-type or unmodified CD80 polypeptide.

In some embodiments, a variant CD80 polypeptide with increased orgreater binding affinity to CD28, PD-L1, and/or CTLA-4 will have anincrease in binding affinity relative to the wild-type or unmodifiedCD80 polypeptide control of at least about 5%, such as at least about10%, 15%, 20%, 25%, 35%, or 50% for the CD28, PD-L1, and/or CTLA-4. Insome embodiments, the increase in binding affinity relative to thewild-type or unmodified CD80 polypeptide is more than 1.2-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold. In such examples,the wild-type or unmodified CD80 polypeptide has the same sequence asthe variant CD80 polypeptide except that it does not contain the one ormore amino acid modifications (e.g., substitutions).

In some embodiments, a variant CD80 polypeptide with decreased orreduced binding affinity to CD28, PD-L1, and/or CTLA-4 will havedecrease in binding affinity relative to the wild-type or unmodifiedCD80 polypeptide control of at least 5%, such as at least about 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more for the CD28, PD-L1,and/or CTLA-4. In some embodiments, the decrease in binding affinityrelative to the wild-type or unmodified CD80 polypeptide is more than1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold. In suchexamples, the wild-type or unmodified CD80 polypeptide has the samesequence as the variant CD80 polypeptide except that it does not containthe one or more amino acid modifications (e.g., substitutions).

In some embodiments, the equilibrium dissociation constant (K_(d)) ofany of the foregoing embodiments to CD28, PD-L1, and/or CTLA-4 can beless than 1×10⁻⁵M, 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹M, 1×10⁻¹⁰ M or1×10⁻¹¹ M, or 1×10⁻¹² M.

The wild-type or unmodified CD80 sequence does not necessarily have tobe used as a starting composition to generate variant CD80 polypeptidesdescribed herein. Therefore, use of the term “substitution” does notimply that the provided embodiments are limited to a particular methodof making variant CD80 polypeptides. Variants CD80 polypeptides can bemade, for example, by de novo peptide synthesis and thus does notnecessarily require a “substitution” in the sense of altering a codon toencode for the substitution. This principle also extends to the terms“addition” and “deletion” of an amino acid residue which likewise do notimply a particular method of making. The means by which the variant CD80polypeptides are designed or created is not limited to any particularmethod. In some embodiments, however, a wild-type or unmodified CD80encoding nucleic acid is mutagenized from wild-type or unmodified CD80genetic material and screened for desired specific binding affinityand/or induction of IFN-gamma expression or other functional activityaccording to the methods disclosed in the Examples or other methodsknown to a skilled artisan. In some embodiments, a variant CD80polypeptide is synthesized de novo utilizing protein or nucleic acidsequences available at any number of publicly available databases andthen subsequently screened. The National Center for BiotechnologyInformation provides such information and its website is publiclyaccessible via the internet as is the UniProtKB database as discussedpreviously.

Unless stated otherwise, as indicated throughout the present disclosure,the amino acid substitution(s) are designated by amino acid positionnumber corresponding to the numbering of positions of the unmodified ECDsequence set forth in SEQ ID NO:28 or also, where applicable, theunmodified IgV sequence set forth in SEQ ID NO:152 or 372 (containingresidues 1-101 or 1-107, respectively, of SEQ ID NO:28 depending onannotation convention) as follows:

(SEQ ID NO: 28) VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTT KQEHFPDN(SEQ ID NO: 152) VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEV T (SEQ ID NO: 372)VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVIQALRPSDEGTYECVVLKYEKDGFKREHLAEV TLSVKAD

It is within the level of a skilled artisan to identify thecorresponding position of a modification, e.g. amino acid substitution,in a CD80 polypeptide, including portion thereof containing an IgSFdomain (e.g. IgV) thereof, such as by alignment of a reference sequencewith SEQ ID NO:28 or SEQ ID NO:152 or SEQ ID NO:372. In the listing ofmodifications throughout this disclosure, the amino acid position isindicated in the middle, with the corresponding unmodified (e.g.wild-type) amino acid listed before the number and the identifiedvariant amino acid substitution listed after the number. If themodification is a deletion of the position a “del” is indicated and ifthe modification is an insertion at the position an “ins” is indicated.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) in a wild-type or unmodifiedCD80 sequence. The one or more amino acid modifications (e.g.,substitutions) can be in the ectodomain (extracellular domain) of thewild-type or unmodified CD80 sequence, such as the extracellular domain.In some embodiments, the one or more amino acid modifications (e.g.,substitutions) are in the IgV domain or specific binding fragmentthereof. In some embodiments, the one or more amino acid modifications(e.g., substitutions) are in the IgC domain or specific binding fragmentthereof. In some embodiments of the variant CD80 polypeptide, some ofthe one or more amino acid modifications (e.g., substitutions) are inthe IgV domain or a specific binding fragment thereof, and some of theone or more amino acid modifications (e.g., substitutions) are in theIgC domain or a specific binding fragment thereof.

In some embodiments, the variant CD80 polypeptide has up to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidmodifications (e.g., substitutions). The modifications (e.g.,substitutions) can be in the IgV domain or the IgC domain. In someembodiments, the variant CD80 polypeptide has up to 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidmodifications (e.g., substitutions) in the IgV domain or specificbinding fragment thereof. In some embodiments, the variant CD80polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 amino acid modifications (e.g., substitutions) inthe IgC domain or specific binding fragment thereof. In someembodiments, the variant CD80 polypeptide has at least about 85%, 86%,86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity with the wild-type or unmodified CD80 polypeptide orspecific binding fragment thereof, such as the amino acid sequence ofSEQ ID NO: 28, 152, or 372.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) in an unmodified CD80 orspecific binding fragment there of corresponding to position(s) 4, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 24, 25, 27, 28, 29, 30,31, 33, 36, 37, 38, 40, 41, 42, 43, 44, 47, 48, 50, 52, 53, 54, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 72, 74, 76, 77, 80, 81, 83, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, 102, 103, 104, 107,108, 109, 110, 114, 115, 116, 117, 118, 120, 121, 122, 126, 127, 128,129, 130, 133, 137, 140, 142, 143, 144, 148, 149, 152, 154, 160, 162,164, 168, 169, 174, 175, 177, 178, 183, 178, 185, 188, 190, 192, 193, or199 with reference to numbering of SEQ ID NO: 28. In some embodiments,such variant CD80 polypeptides exhibit altered binding affinity to oneor more of CD28, PD-L1, or CTLA-4 compared to the wild-type orunmodified CD80 polypeptide. For example, in some embodiments, thevariant CD80 polypeptide exhibits increased binding affinity to CD28,PD-L1, and/or CTLA-4 compared to a wild-type or unmodified CD80polypeptide. In some embodiments, the variant CD80 polypeptide exhibitsdecreased binding affinity to CD28, PD-L1, and/or CTLA-4 compared to awild-type or unmodified CD80 polypeptide.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid substitution selected from V4M, K9E, E10R, V11S, A12G, A12T, A12V,T13N, L14A, S15V, S15F, C16S, C16G, C16L, G17W, H18L, H18R, H18Y, V20L,S21P, V22A, E24G, L25P, Q27R, T28A, T28S, R29C, R29D, R29H, R29V, I30V,Y31F, Y31H, Y31L, Q33H, K36E, K36G, K37E, K37Q, M38I, M38L, M38T, M38V,L40M, T41A, T41G, T41D, T41I, M42T, M43I, M43Q, M43R, M43V, S44P, M47T,N48D, N48I, W50G, E52G, Y53C, K54M, F59L, F59S, D60V, I61N, T62S, N63S,N64S, L65H, S66H, I67F, I67T, V68A, V68M, I69T, L70Q, L70P, L70R, L72P,P74L, D76G, E77G, E77K, Y80N, E81A, E81R, E81V, V83A, V83I, L85I, L85R,K86E, Y87N, E88D, E88G, K89E, K89N, K89R, D90K, D90L, D90N, A91E, A91G,A91S, A91T, F92L, F92N, F92P, F92Y, K93I, K93E, K93Q, K93R, K93V, R94G,R94L, R94F, E95K, H96R, L97R, E99D, E99G, L102S, S103L, S103P, V104A,V104L, D107N, F108L, P109S, P109H, T110A, S114T, D115G, F116S, F116L,E117V, E117G, I118V, I118A, I118T, T120S, S121P, N122S, I126L, I126V,I127T, C128Y, C128R, S129L, S129P, T130A, G133D, P137L, S140T, L142S,E143G, N144D, N144S, L148S, N149D, N149S, N152T, T154I, T154A, E160G,E162G, Y164H, S168G, K169E, K169I, K169S, M174T, M174V, T175A, N177S,H178R, L183H, K185E, H188D, H188Q, R190S, N192D, Q193L, or T199S. Insome embodiments, the variant CD80 polypeptide has one or more aminoacid substitutions selected from the group consisting of V4M, K9E, E10R,V11S, A12G, A12T, A12V, T13N, L14A, S15V, S15F, C16S, C16G, C16L, G17W,H18L, H18R, H18Y, V20L, S21P, V22A, E24G, L25P, Q27R, T28A, T28S, R29C,R29D, R29H, R29V, I30V, Y31F, Y31H, Y31L, Q33H, K36E, K36G, K37E, K37Q,M38I, M38L, M38T, M38V, L40M, T41A, T41G, T41D, I41I, M42T, M43I, M43Q,M43R, M43V, S44P, M47T, N48D, N48I, W50G, E52G, Y53C, K54M, F59L, F59S,D60V, I61N, T62S, N63S, N64S, L65H, S66H, I67F, 167T, V68A, V68M, 169T,L70Q, L70P, L70R, L72P, P74L, D76G, E77G, E77K, Y80N, E81A, E81R, E81V,V83A, V83I, L85I, L85R, K86E, Y87N, E88D, E88G, K89E, K89N, K89R, D90K,D90L, D90N, A91E, A91G, A91S, A91T, F92L, F92N, F92P, F92Y, K93I, K93E,K93Q, K93R, K93V, R94G, R94L, R94F, E95K, H96R, L97R, E99D, E99G, L102S,S103L, S103P, V104A, V104L, D107N, F108L, P109S, P109H, T110A, S114T,D115G, F116S, F116L, E117V, E117G, I118V, I118A, I118T, T120S, S121P,N122S, I126L, I126V, I127T, C128Y, C128R, S129L, S129P, T130A, G133D,P137L, S140T, L142S, E143G, N144D, N144S, L148S, N149D, N149S, N152T,T154I, T154A, E160G, E162G, Y164H, S168G, K169E, K169I, K169S, M174T,M174V, T175A, N177S, H178R, L183H, K185E, H188D, H188Q, R190S, N192D,Q193L, T199S or a conservative amino acid substitution thereof.

In some embodiments, the one or more amino acid modification, e.g.substitution is L70Q/A91G, L70Q/A91G/T130A, L70Q/A91G/I118A/T120S/T130A,V4M/L70Q/A91G/T120S/T130A, L70Q/A91G/T120S/T130A,V20L/L70Q/A91S/T120S/T130A, S44P/L70Q/A91G/T130A,L70Q/A91G/E117G/T120S/T130A, A91G/T120S/T130A, L70R/A91G/T120S/T130A,L70Q/E81A/A91G/T120S/I127T/T130A, L70Q/Y87N/A91G/T130A,T28S/L70Q/A91G/E95K/T120S/T130A, N63S/L70Q/A91G/T120S/T130A,K36E/I67T/L70Q/A91G/T120S/T130A/N152T, E52G/L70Q/A91G/T120S/T130A,K37E/F59S/L70Q/A91G/T120S/T130A, A91G/S103P, K89E/T130A,D60V/A91G/T120S/T130A, K54M/A91G/T120S,M38T/L70Q/E77G/A91G/T120S/T130A/N152T, R29H/E52G/L70R/E88G/A91G/T130A,Y31H/T41G/L70Q/A91G/T120S/T130A, V68A/T110A, S66H/D90G/T110A/F116L,R29H/E52G/T120S/T130A, A91G/L102S, I67T/L70Q/A91G/T120S,L70Q/A91G/T110A/T120S/T130A,M38V/T41D/M43I/W50G/D76GN83A/K89E/T120S/T130A, V22A/L70Q/S121P,A12V/S15F/Y31H/T41G/T130A/P137L/N152T, I67F/L70R/E88G/A91G/T120S/T130A,E24G/L25P/L70Q/T120S, A91G/F92L/F108L/T120S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,E24G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/H96R/N149S/C182S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29V/M43Q/E81R/L85I/K89R/D90L/A91E/F92N/K93Q/R94G, T41I/A91G,K89R/D90K/A91G/F92Y/K93R/N122S/N177S, K89R/D90K/A91G/F92Y/K93R,K36G/K37Q/M38I/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/E99G/T130A/N149S,E88D/K89R/D90K/A91G/F92Y/K93R, K36G/K37Q/M38I/L40M,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S,A12T/H18L/M43V/F59L/E77K/P109S/I118T,R29V/Y31F/K36G/M38L/M43Q/E81R/V83I/L85I/K89R/D90L/A91E/F92N/K93Q/R94G,V68M/L70P/L72P/K86E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/M174T,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/H188D,H18R/R29D/Y31L/Q33H/K36G/K37E/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/E143G/K169E/M174V/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/F108L/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/C128Y/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/T130A/K169E,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93I/R94L/L97R/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93I/R94L/L97R/T130A/L148S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/I61N/E81V/L85R/K89N/A91T/F92P/K93V/R94F/V104A/T120S/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/F92P/K93V/R94F/I118V/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/T175A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/F116S/T130A/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/L142S/H188D,C16S/H18L/R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T110A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/A91G/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/L70Q/D76G/A91G/S103L/T120S/I127T/T130A,DELTAQ33/Y53C/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/S129L/H188D,K9E/E10R/V11S/A12G/T13N/K14A/S15V/C16L/G17W/H18Y/Y53C/L70Q/D90G/T130A/N149D/N152T/H188D,H18L/R29D/Y31L/Q33H/K36G/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,K89E/K93E/T130A,S21P/R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/N48I/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/P109H/I126L/K169I,H18L/R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/P74L/Y80N/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R,S21P/R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/P74L/Y80N/E81V/L85R/K89N/D90N/A91T/F92P/K93V/R94L/T130A/N149S/E162G,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/R190S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/P74L/Y80N/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/R190S,C16GN22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/D76G/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169I/H178R/N192D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/E117V/I118T/N149S/S168G/H188Q,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N64S/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118T/T130A/N149S/K169I,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/D115G/I118T/T130A/G133D/N149S,S129P, A91G/S129P, I69T/L70Q/A91G/T120S, Y31H/S129P,T28A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/V104L/T130A/N149S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/N149S/H188Q,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/N149S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/T154I,A12G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/T130A/L183H,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I/Q193L,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/T130A/N149S/K169I,I118T/C128R, Q27R/R29C/M42T/S129P/E160G, S129P/T154A,S21P/L70Q/D90G/T120S/T130A, L70Q/A91G/N144D,L70Q/A91G/I118A/T120S/T130A/K169E,V4M/L70Q/A91G/I118V/T120S/T130A/K169E,L70Q/A91G/I118V/T120S/T130A/K169E, L70Q/A91G/I118V/T120S/T130A,V20L/L70Q/A91S/I118V/T120S/T130A, L70Q/A91G/E117G/I118V/T120S/T130A,A91G/I118V/T120S/T130A, L70R/A91G/I118V/T120S/T130A/T199S,L70Q/E81A/A91G/I118V/T120S/I127T/T130A,T28S/L70Q/A91G/E95K/I118V/T120S/I126V/T130A/K169E,N63S/L70Q/A91G/S114T/I118V/T120S/T130A,K36E/I67T/L70Q/A91G/I118V/T120S/T130A/N152T,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E,K37E/F59S/L70Q/A91G/I118V/T120S/T130A/K185E,D60V/A91G/I118V/T120S/T130AK169E, K54M/L70Q/A91G/Y164H/T120S,M38T/L70Q/E77G/A91G/I118V/T120S/T130A/N152T,Y31H/T41G/M43L/L70Q/A91G/I118V/T120S/I126V/T130A, L65H/D90G/T110A/F116L,R29H/E52G/D90N/I118V/T120S/T130A, I67T/L70Q/A91G/I118V/T120S,L70Q/A91G/T110A/I118V/T120S/T130A,M38V/T41D/M43I/W50G/D76GN83A/K89E/I118V/T120S/I126V/T130A,A12V/S15F/Y31H/M38L/T41G/M43L/D90N/T130A/P137L/N149D/N152T,I67F/L70R/E88G/A91G/I118V/T120S/T130A,E24G/L25P/L70Q/A91G/I118V/T120S/N152T, A91G/F92L/F108L/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S,K36G/K37Q/M38I/L40M/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/E99G/T130A/N149S,K36G/L40M,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/M174T,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N48D/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,H18R/R29D/Y31L/Q33H/K36G/K37E/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/E143G/K169E/M174V/H188D,R29D/I30V/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/F108L/I118V/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/N149D/K169E/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/C128Y/T130A/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/E99D/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/I61N/E81V/L85R/K89N/A91T/F92P/K93V/R94F/V104A/I118V/T120S/I126V/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118V/T120S/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/T175A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/L1425/H188D,C16S/H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T110A/I118V/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/A91G/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/D76G/A91G/S103L/I118V/T120S/I127T/T130A,Y53C/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E,Y53C/L70Q/D90G/T130A/N149D/N152T/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) in an unmodified CD80 orspecific binding fragment there of corresponding to position(s) 4, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 24, 25, 27, 28, 29, 31,33, 36, 37, 38, 40, 41, 42, 43, 44, 47, 48, 50, 52, 53, 54, 59, 60, 61,62, 63, 64, 66, 67, 68, 69, 70, 72, 74, 76, 77, 80, 81, 83, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, 102, 103, 104, 108, 109,110, 115, 116, 117, 118, 120, 121, 122, 126, 127, 128, 129, 130, 133,137, 140, 142, 143, 144, 148, 149, 152, 154, 160, 162, 168, 169, 174,175, 177, 178, 183, 178, 188, 190, 192, or 193 with reference tonumbering of SEQ ID NO: 28. In some embodiments, such variant CD80polypeptides exhibit altered binding affinity to one or more of CD28,PD-L1, or CTLA-4 compared to the wild-type or unmodified CD80polypeptide. For example, in some embodiments, the variant CD80polypeptide exhibits increased binding affinity to CD28, PD-L1, and/orCTLA-4 compared to a wild-type or unmodified CD80 polypeptide. In someembodiments, the variant CD80 polypeptide exhibits decreased bindingaffinity to CD28, PD-L1, and/or CTLA-4 compared to a wild-type orunmodified CD80 polypeptide.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid substitution selected from V4M, K9E, E10R, V11S, A12G, A12T, A12V,T13N, L14A, S15V, S15F, C16S, C16G, C16L, G17W, H18L, H18R, H18Y, V20L,S21P, V22A, E24G, L25P, Q27R, T28A, T28S, R29C, R29D, R29H, R29V, Y31F,Y31H, Y31L, Q33H, K36E, K36G, K37E, K37Q, M38I, M38L, M38T, M38V, L40M,T41A, T41G, T41D, T41I, M42T, M43I, M43Q, M43R, M43V, S44P, M47T, N48I,W50G, E52G, Y53C, K54M, F59L, F59S, D60V, I61N, T62S, N63S, N64S, S66H,I67F, I67T, V68A, V68M, I69T, L70Q, L70P, L70R, L72P, P74L, D76G, E77G,E77K, Y80N, E81A, E81R, E81V, V83A, V83I, L85I, L85R, K86E, Y87N, E88D,E88G, K89E, K89N, K89R, D90K, D90L, D90N, A91E, A91G, A91S, A91T, F92L,F92N, F92P, F92Y, K93I, K93E, K93Q, K93R, K93V, R94G, R94L, R94F, E95K,H96R, L97R, E99G, L102S, S103L, S103P, V104A, V104L, F108L, P109S,P109H, T110A, D115G, F116S, F116L, E117V, E117G, I118V, I118A, I118T,T120S, S121P, N122S, I126L, I127T, C128Y, C128R, S129L, S129P, T130A,G133D, P137L, S140T, L142S, E143G, N144S, L148S, N149D, N149S, N152T,T154I, T154A, E160G, E162G, S168G, K169E, K169I, K169S, M174T, M174V,T175A, N177S, H178R, L183H, H188D, H188Q, R190S, N192D, Q193L. In someembodiments, the variant CD80 polypeptide has one or more amino acidsubstitutions selected from the group consisting of V4M, K9E, E10R,V11S, A12G, A12T, A12V, T13N, L14A, S15V, S15F, C16S, C16G, C16L, G17W,H18L, H18R, H18Y, V20L, S21P, V22A, E24G, L25P, Q27R, T28A, T28S, R29C,R29D, R29H, R29V, Y31F, Y31H, Y31L, Q33H, K36E, K36G, K37E, K37Q, M38I,M38L, M38T, M38V, L40M, T41A, T41G, T41D, T41I, M42T, M43I, M43Q, M43R,M43V, S44P, M47T, N48I, W50G, E52G, Y53C, K54M, F59L, F59S, D60V, I61N,T62S, N63S, N64S, S66H, I67F, 167T, V68A, V68M, 169T, L70Q, L70P, L70R,L72P, P74L, D76G, E77G, E77K, Y80N, E81A, E81R, E81V, V83A, V83I, L85I,L85R, K86E, Y87N, E88D, E88G, K89E, K89N, K89R, D90K, D90L, D90N, A91E,A91G, A91S, A91T, F92L, F92N, F92P, F92Y, K93I, K93E, K93Q, K93R, K93V,R94G, R94L, R94F, E95K, H96R, L97R, E99G, L102S, S103L, S103P, V104A,V104L, F108L, P109S, P109H, T110A, D115G, F116S, F116L, E117V, E117G,I118V, I118A, I118T, T120S, S121P, N122S, I126L, I127T, C128Y, C128R,S129L, S129P, T130A, G133D, P137L, S140T, L142S, E143G, N144S, L148S,N149D, N149S, N152T, T154I, T154A, E160G, E162G, S168G, K169E, K169I,K169S, M174T, M174V, T175A, H178R, H178R, L183H, H188D, H188Q, R190S,N192D, Q193L, or a conservative amino acid substitution thereof.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) in an unmodified CD80 orspecific binding fragment there of corresponding to position(s) 30, 65,107, 114, 164, 185, or 199 with reference to numbering of SEQ ID NO: 28.In some embodiments, the variant CD80 polypeptide has one or more aminoacid substitution selected from I30V, N48D, L65H, E99D, D107N, S114T,I126V, N144D, Y164H, and T199S. In some embodiments, the variant CD80polypeptide has one or more amino acid substitutions selected from thegroup consisting of N48D, L65H, E99D, D107N, S114T, I126V, N144D, Y164H,K185E, T199S, or a conservative amino acid substitution thereof.

A conservative amino acid substitution is any amino acid that falls inthe same class of amino acids as the substituted amino acids, other thanthe wild-type or unmodified amino acid. The classes of amino acids arealiphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxylor sulfur-containing (serine, cysteine, threonine, and methionine),cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic(histidine, lysine, and arginine), and acidic/amide (aspartate,glutamate, asparagine, and glutamine). Thus, for example, a conservativeamino acid substitution of the V4M substitution includes V4S, V4C, andV4T amino acid substitutions.

In some embodiments, the variant CD80 polypeptide comprises any of thesubstitutions (mutations) listed in Table 1. Table 1 also providesexemplary sequences by reference to SEQ ID NO for the extracellulardomain (ECD) or IgV domain of wild-type CD80 or exemplary variant CD80polypeptides. As indicated, the exact locus or residues corresponding toa given domain can vary, such as depending on the methods used toidentify or classify the domain. Also, in some cases, adjacent N- and/orC-terminal amino acids of a given domain (e.g. IgV) also can be includedin a sequence of a variant IgSF polypeptide, such as to ensure properfolding of the domain when expressed. Thus, it is understood that theexemplification of the SEQ ID NOs in Table 1 is not to be construed aslimiting. For example, the particular domain, such as the IgV domain, ofa variant CD80 polypeptide can be several amino acids longer or shorter,such as 1-10, e.g. 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter,than the sequence of amino acids set forth in the respective SEQ ID NO.

In some embodiments, the variant CD80 polypeptide comprises any of theextracellular domain (ECD) sequences listed in Table 1 (i.e., any one ofSEQ ID NOS: 55-108, 280-346, 414-475). In some embodiments, the variantCD80 polypeptide comprises a polypeptide sequence that exhibits at least90% identity, at least 91% identity, at least 92% identity, at least 93%identity, at least 94% identity, at least 95% identity, such as at least96% identity, 97% identity, 98% identity, or 99% identity to any of theextracellular domain (ECD) sequences listed in Table 1 (i.e., any one ofSEQ ID NOS: 55-108, 280-346, 414-475) and contains the amino acidmodification(s), e.g., substitution(s), not present in the wild-type orunmodified CD80. In some embodiments, the variant CD80 polypeptidecomprises a specific binding fragment of any of the extracellular domain(ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 55-108,280-346, 414-475) and contains the amino acid modification(s), e.g.,substitution(s), not present in the wild-type or unmodified CD80. Insome embodiments, the variant CD80 polypeptide comprises any of the IgVsequences listed in Table 1 (i.e., any one of SEQ ID NOS: 153-195, 347,373-386, 476-477). In some embodiments, the variant CD80 polypeptidecomprises a polypeptide sequence that exhibits at least 90% identity, atleast 91% identity, at least 92% identity, at least 93% identity, atleast 94% identity, at least 95% identity, such as at least 96%identity, 97% identity, 98% identity, or 99% identity to any of the IgVsequences listed in Table 1 (i.e., any one of SEQ ID NOS: 153-195, 347,373-386, 476-477) and contains the amino acid modification(s), e.g.,substitution(s), not present in the wild-type or unmodified CD80. Insome embodiments, the variant CD80 polypeptide comprises a specificbinding fragment of any of the IgV sequences listed in Table 1 (i.e.,any one of SEQ ID NOS: 153-195, 347, 373-386, 476-477) and contains theamino acid modification(s), e.g., substitution(s), not present in thewild-type or unmodified CD80.

Table 1 also provides exemplary sequences by reference to SEQ ID NO forthe extracellular domain (ECD) or IgV domain of wild-type CD80 orexemplary variant CD80 polypeptides. As indicated, the exact locus orresidues corresponding to a given domain can vary, such as depending onthe methods used to identify or classify the domain. Also, in somecases, adjacent N- and/or C-terminal amino acids of a given domain (e.g.ECD) also can be included in a sequence of a variant IgSF polypeptide,such as to ensure proper folding of the domain when expressed. Thus, itis understood that the exemplification of the SEQ ID NOSs in Table 1 isnot to be construed as limiting. For example, the particular domain,such as the IgV domain, of a variant CD80 polypeptide can be severalamino acids longer or shorter, such as 1-10, e.g. 1, 2, 3, 4, 5, 6 or 7amino acids longer or shorter, than the sequence of amino acids setforth in the respective SEQ ID NO.

TABLE 1 Exemplary variant CD80 polypeptides ECD SEQ IgV ID SEQMutation(s) NO ID NO Wild-type 28 152, 372 L70Q/A91G 55 153, 374L70Q/A91G/T130A 56 L70Q/A91G/I118A/T120S/T130A 57V4M/L70Q/A91G/T120S/T130A 58 154 L70Q/A91G/T120S/T130A 59V20L/L70Q/A91S/T120S/T130A 60 155 S44P/L70Q/A91G/T130A 61 156L70Q/A91G/E117G/T120S/T130A 62 A91G/T120S/T130A 63 157L70R/A91G/T120S/T130A 64 158 L70Q/E81A/A91G/T120S/I127T/T130A 65 159L70Q/Y87N/A91G/T130A 66 160 T28S/L70Q/A91G/E95K/T120S/T130A 67 161N63S/L70Q/A91G/T120S/T130A 68 162 K36E/I67T/L70Q/A91G/T120S/T130A/N152T69 163 E52G/L70Q/A91G/T120S/T130A 70 164 K37E/F595/L70Q/A91G/T120S/T130A71 165 A91G/S103P 72 378 K89E/T130A 73 166, 385 A91G 74 154, 375D60V/A91G/T120S/T130A 75 167 K54M/A91G/T120S 76 168M38T/L70Q/E77G/A91G/T120S/T130A/N152T 77 169R29H/E52G/L70R/E88G/A91G/T130A 78 170 Y31H/T41G/L70Q/A91G/T120S/T130A 79171 V68A/T110A 80 172 S66H/D90G/T110A/F116L 81 173 R29H/E52G/T120S/T130A82 174 A91G/L102S 83 386 I67T/L70Q/A91G/T120S 84 175L70Q/A91G/T110A/T120S/T130A 85M38V/T41D/M43I/W50G/D76G/V83A/K89E/T120S/T130A 86 176 V22A/L70Q/S121P 87177 A12V/S15F/Y31H/T41G/T130A/P137L/N152T 88 178I67F/L70R/E88G/A91G/T120S/T130A 89 179 E24G/L25P/L70Q/T120S 90 180A91G/F92L/F108L/T120S 91 181R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 92 182A91T/F92P/K93V/R94L/I118T/N149SR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 93A91T/F92P/K93V/R94L/N144S/N149SR29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/ 94 183K89N/A91T/F92P/K93V/R94L/L148S/N149SE24G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/F59L/E81V/ 95 184L85R/K89N/A91T/F92P/K93V/R94L/H96R/N149S/C182SR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 96A91T/F92P/K93V/R94L/N149SR29V/M43Q/E81R/L85I/K89R/D90L/A91E/F92N/K93Q/R94G 97 185, 376 T41I/A91G98 186, 377 K89R/D90K/A91G/F92Y/K93R/N122S/N177S 99K89R/D90K/A91G/F92Y/K93R 100 187, 373K36G/K37Q/M38I/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/ 101 188E99G/T130A/N149S E88D/K89R/D90K/A91G/F92Y/K93R 102 189, 379K36G/K37Q/M38I/L40M 103 190, 380 K36G 104 191, 381R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S 105 192A12T/H18L/M43V/F59L/E77K/P109S/I118T 106 193R29V/Y31F/K36G/M38L/M43Q/E81R/V83I/L85I/K89R/D90L/A91E/ 107 194, 382F92N/K93Q/R94G V68M/L70P/L72P/K86E 108 195, 383R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 280A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169ER29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 281K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130AH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 282K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169ER29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 283A91T/F92P/K93V/R94L/T120S/T130A/M174TR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/F59L/E81V/L85R/ 284K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/H188DH18R/R29D/Y31L/Q33H/K36G/K37E/M38I/T41A/M43R/M47T/L70Q/ 285E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 286K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/E143G/K169E/ M174V/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 287K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 288A91T/F92P/K93V/R94L/T120S/I127T/T130A/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 289K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169ER29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 290K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L85R/K89N/A91T/ 291F92P/K93V/R94L/T120S/I127T/T130A/K169E/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 292A91T/F92P/K93V/R94L/F108L/T120S/T130A/K169E/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 293K89N/A91T/F92P/K93V/R94L/T130A/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 294K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169EH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/ 295L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 296A91T/F92P/K93V/R94L/T120S/I127T/C128Y/T130A/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 297A91T/F92P/K93V/R94F/T130A/K169EH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 298K89N/A91T/F92P/K93V/R94L/T130AH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/ 299L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169ER29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 300K89N/A91T/F92P/K931/R94L/L97R/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 301K89N/A91T/F92P/K931/R94L/L97R/T130A/L148SH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 302K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169ER29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/I61N/E81V/L85R/ 303K89N/A91T/F92P/K93V/R94F/V104A/T120S/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 304K89N/F92P/K93V/R94F/I118V/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/T62S/E81V/L85R/ 305K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/T175AH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 306K89N/A91T/F92P/K93V/R94L/F116S/T130A/H188DH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 307K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/L142S/H188DC16S/H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/ 308L85R/K89N/A91T/F92P/K93V/R94L/T110A/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/A91G/T120S/ 309I127T/T130A/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/D76G/A91G/ 310S103L/T120S/I127T/T130ADELTAQ33/Y53C/L85R/K89N/A91T/F92P/K93V/R94L/T120S/ 311 I127T/T130A/K169ET62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E 312R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 313K89N/A91T/F92P/K93V/R94L/S129L/H188DK9E/E10R/V11S/A12G/T13N/K14A/S15V/C16L/G17W/H18Y/Y53C/ 314L70Q/D90G/T130A/N149D/N152T/H188DH18L/R29D/Y31L/Q33H/K36G/T41A/M43R/M47T/E81V/L85R/ 315K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D K89E/K93E/T130A316 S21P/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/ 317N48I/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/ P109H/I126L/K169IH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/ 318 347, 384P74L/Y80N/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97RS21P/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/P74L/Y80N/ 319E81V/L85R/K89N/D90N/A91T/F92P/K93V/R94L/T130A/N149S/E162GH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/ 320L85R/K89N/A91T/F92P/K93V/R94L/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/ 321K89N/A91T/F92P/K93V/R94L/T130A/N149S/R190SH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/P74L/Y80N/ 322E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/R190SC16G/V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/ 323D76G/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169I/H178R/N192DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 324A91T/F92P/K93V/R94F/E117V/I118T/N149S/S168G/H188QV22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/ 325L85R/K89N/A91T/F92P/K93V/R94L/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N64S/E81V/ 326L85R/K89N/A91T/F92P/K93V/R94F/I118T/T130A/N149S/K169IV22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/ 327L85R/K89N/A91T/F92P/K93V/R94L/D115G/I118T/T130A/G133D/N149S S129P 328A91G/S129P 329 I69T/L70Q/A91G/T120S 330 Y31H/S129P 331T28A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 332K89N/A91T/F92P/K93V/R94L/V104L/T130A/N149SH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 333K89N/A91T/F92P/K93V/R94L/L97R/N149S/H188QH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 334K89N/A91T/F92P/K93V/R94L/L97R/N149SH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/ 335L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/T154IA12G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/ 336L85R/K89N/A91T/F92P/K93V/R94L/L97R/T130A/L183HR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 337A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169SR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 338A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I/Q193LV22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/ 339L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149SR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 340K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149SR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 341K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169IR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 342K89N/A91T/F92P/K93V/R94F/T130A/N149S/K169I I118T/C128R 343Q27R/R29C/M42T/S129P/E160G 344 S129P/T154A 345S21P/L70Q/D90G/T120S/T130A 346 L70Q/A91G/N144D 414L70Q/A91G/I118A/T120S/T130A/K169E 415V4M/L70Q/A91G/I118V/T120S/T130A/K169E 416L70Q/A91G/I118V/T120S/T130A/K169E 417 L70Q/A91G/I118V/T120S/T130A 418V20L/L70Q/A91S/I118V/T120S/T130A 419 L70Q/A91G/E117G/I118V/T120S/T130A420 A91G/I118V/T120S/T130A 421 L70R/A91G/I118V/T120S/T130A/T199S 422L70Q/E81A/A91G/I118V/T120S/I127T/T130A 423T28S/L70Q/A91G/E95K/I118V/T120S/I126V/T130A/K169E 424N63S/L70Q/A91G/S114T/I118V/T120S/T130A 425K36E/I67T/L70Q/A91G/I118V/T120S/T130A/N152T 426E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E 427K37E/F59S/L70Q/A91G/I118V/T120S/T130A/K185E 428D60V/A91G/I118V/T120S/T130A/K169E 429 K54M/L70Q/A91G/Y164H/T120S 430M38T/L70Q/E77G/A91G/I118V/T120S/T130A/N152T 431Y31H/T41G/M43L/L70Q/A91G/I118V/T120S/I126V/T130A 432L65H/D90G/T110A/F116L 433 R29H/E52G/D90N/I118V/T120S/T130A 434I67T/L70Q/A91G/I118V/T120S 436 L70Q/A91G/T110A/I118V/T120S/T130A 437M38V/T41D/M43I/W50G/D76G/V83A/K89E/I118V/T120S/ 438 I126V/T130AA12V/S15F/Y31H/M38L/T41G/M43L/D90N/T130A/P137L/ 439 N149D/N152TI67F/L70R/E88G/A91G/I118V/T120S/T130A 440E24G/L25P/L70Q/A91G/I118V/T120S/N152T 441 A91G/F92L/F108L/I118V/T120S442 E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S 443K36G/K37Q/M38I/L40M/F59L/E81V/L85R/K89N/A91T/F92P/ 444K93V/R94L/E99G/T130A/N149S K36G/L40M 445 476, 477R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 446K89N/A91T/F92P/K93V/R94L/I118V/T120S/ I127T/T130A/K169ER29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/ 447L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/ I127T/T130AH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/ 448L85R/K89N/A91T/F92P/K93V/R94L/I118V/ T120S/I127T/T130A/K169ER29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 449K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/ K169E/M174TR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N48D/F59L/ 450E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/ I127T/T130A/H188DH18R/R29D/Y31L/Q33H/K36G/K37E/M38I/T41A/M43R/M47T/L70Q/ 451E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/ K169E/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 452K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/E143G/K169E/M174V/H188D R29D/I30V/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 453K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 454A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 455K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169ER29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/K89N/ 456A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L85R/K89N/A91T/ 457F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 458A91T/F92P/K93V/R94L/F108L/I118V/T120S/T130A/K169E/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/ 459K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/N149D/K169E/ H188DH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/ 460L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 461A91T/F92P/K93V/R94L/I118V/T120S/I127T/C128Y/T130A/H188DH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 462K89N/A91T/F92P/K93V/R94L/E99D/T130AH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/ 463L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169ER29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/I61N/E81V/L85R/ 464K89N/A91T/F92P/K93V/R94F/V104A/I118V/T120S/I126V/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/ 465A91T/F92P/K93V/R94F/I118V/T120S/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/T62S/E81V/L85R/ 466K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/T175AH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/ 467K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/L142S/H188DC16S/H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/ 468L85R/K89N/A91T/F92P/K93V/R94L/T110A/I118V/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/A91G/I118V/T120S/ 469I127T/T130A/H188DR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/D76G/A91G/ 470S103L/I118V/T120S/I127T/T130AY53C/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/ 471 I127T/T130A/K169ET62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/ 472 T120S/T130A/K169EY53C/L70Q/D90G/T130A/N149D/N152T/H188D 473H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/ 474L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/ T130A/H188DH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/ 475L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S

In some embodiments the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) corresponding to position(s)12, 18, 20, 29, 31, 36, 40, 41, 43, 52, 59, 60, 63, 67, 70, 77, 81, 87,88, 89, 90, 91, 92, 93, 107, 109, 114, 117, 118, 120, 122, 127, 130,144, 169, 178 or 199 of SEQ ID NO: 28. In some embodiments, the variantCD80 polypeptide has one or more amino acid substitutions selected fromA12T, H18L, V20L, R29H, Y31H, K36G, L40M, T41G, T41I, M43V, E52G, F59L,D60V, N63S, I67T, L70Q, L70R, E77K, E81A, Y87N, E88D, E88G, K89E, K89R,D90K, D90N, A91G, A91S, F92Y, K93R, D107N, P109S, S114T, E117G, I118A,I118T, I118V, T120S, I127T, T130A, N144D, K169E, H178R, or T199S. Insome embodiments, the variant CD80 polypeptide has one or more aminoacid substitutions selected from A12T, H18L, V20L, R29H, Y31H, K36G,L40M, T41G, T41I, M43V, E52G, F59L, D60V, N63S, I67T, L70Q, L70R, E77K,E81A, Y87N, E88D, E88G, K89E, K89R, D90K, D90N, A91G, A91S, F92Y, K93R,D107N, P109S, S114T, E117G, I118A, I118T, I118V, T120S, I127T, T130A,N144D, K169E, H178R, or T199S or conservative amino acid substitutionsthereof. In some embodiments, the one or more amino acid substitutionsis A12T/H18L/M43V/F59L/E77K/P 109S/I118T, V20L/L70Q/A91S/T120S/T130A,V20L/L70Q/A91S/I118V/T120S/T130A,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,T41I/A91G, E52G/L70/A91G/T120S/T130A,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E, D60V/A91G/T120S/T130A,D60V/A91G/I118V/T120S/T130A/K169E, N63S/L70Q/A91G/T120S/T130A,N63S/L70Q/A91G/S114T/I118V/T120S/T130A, I67T/L70Q/A91G/T120S,I67T/L70Q/A91G/I118V/T120S, L70Q/E81A/A91G/T120S/I127T/T130A,L70Q/E81A/A91G/I118V/T120S/I127T/T130A, L70Q/Y87N/A91G/T130A, L70Q/A91G,L70Q/A91G/N144D, L70Q/A91G/E117G/T120S/T130A,L70Q/A91G/E117G/I118V/T120S/T130A, L70Q/A91G/I118A/T120S/T130A,L70Q/A91G/I118A/T120S/T130A/K169E, L70Q/A91G/T120S/T130A,L70Q/A91G/I118V/T120S/T130A/K169E, L70R/A91G/T120S/T130A,L70R/A91G/I118V/T120S/T130A/T199S, E88D/K89R/D90K/A91G/F92Y/K93R,K89R/D90K/A91G/F92Y/K93R, or K89R/D90K/A91G/F92Y/K93R/N122S/N177S,E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S. In some embodiments, thevariant CD80 polypeptide exhibits increased affinity for the ectodomainof CD28 and/or increased selectivity to CD28 compared to a wild-type orunmodified CD80 polypeptide, such as comprising the sequence set forthin SEQ ID NO: 28, 152, or 372.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) that correspond to position(s)12, 18, 21, 22, 28, 29, 31, 33, 36, 38, 40, 41, 42, 43, 47, 48, 59, 64,67, 68, 70, 77, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 97, 104, 109,115, 117, 118, 120, 122, 126, 130, 133, 140, 144, 148, 149, 168, 169,177, 183, 188 or 193. of SEQ ID NO: 28. In some embodiments, the variantCD80 polypeptide has one or more amino acid substitutions selected fromthe group consisting of A12G, A12T, H18L, S21P, V22A, T28A, R29D, R29H,Y31H, Y31L, Q33H, K36G, M38I, L40M, T41A, T41G, M42T, M43R, M43V, M47T,N48I, F59L, N64S, I67T, V68A, V68M, L70Q, E77K, E81V, L85R, Y87N, E88D,E88G, K89E, K89N, K89R, D90K, D90N, A91G, A91T, F92P, F92Y, K93R, K93V,R94F, R94L, L97R, V104L, P109H, P109S, D115G, E117V, I118T, I118V,T120S, N122S, I126L, T130A, G133D, 5140T, N144S, L148S, N149S, S168G,K169I, K169S, N177S, L183H, H188Q, R190S and Q193L. In some embodiments,the variant CD80 polypeptide has one or more amino acid substitutionsselected from the group consisting of A12T, H18L, R29D, R29H, Y31H,Y31L, Q33H, K36G, M38I, L40M, T41A, T41G, M42T, M43R, M43V, M47T, F59L,I67T, L70Q, E77K, E81V, L85R, Y87N, E88D, E88G, K89E, K89N, K89R, D90K,D90N, A91G, A91T, F92P, F92Y, K93R, K93V, R94L, P109S, I118T, I118V,T120S, N122S, N144S, L148S, N149S, and N178S, and conservative aminoacid substitutions thereof.

In some embodiments, the one or more amino acid substitutions isA12T/H18L/M43V/F59L/E77K/P109S/I118T,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R, K89R/D90K/A91G/F92Y/K93R, A91G,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/R190S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/E117V/I118T/N149S/S168G/H188Q,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/D115G/I118T/T130A/G133D/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N64S/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118T/T130A/N149S/K169I,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/D115G/I118T/T130A/G133D/N149S,T28A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/V104L/T130A/N149S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/N149S/H188Q,K89E/T130A, K89E/K93E/T130A,S21P/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N48I/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/S21P/N48I/V68A/P109H/I126L/K169I,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S,A12G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/T130A/L183H,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I/Q193L,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I, orR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/T130A/N149S/K169I.In some embodiments, the variant CD80 polypeptide exhibits increasedaffinity to PD-L1 and/or increased selectivity to PD-L1 compared to thewild-type or unmodified CD80 polypeptide, such as comprising thesequence set forth in SEQ ID NO: 28, 152, or 372.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) that correspond to position(s)12, 18, 36, 40, 43, 59, 77, 88, 89, 90, 91, 92, 93, 109, 118, 122, or177 of SEQ ID NO: 28. In some embodiments, the variant CD80 polypeptidehas one or more amino acid substitutions selected from the groupconsisting of A12T, H18L, K36G, M43V, F59L, E77K, E88D, K89R, D90K,A91G, F92Y, K93R, P109S, I118T, N112S, and N177S. In some embodiments,the variant CD80 polypeptide has one or more amino acid substitutionsselected from the group consisting of A12T, H18L, K36G, L40M, M43V,F59L, E77K, E88D, K89R, D90K, A91G, F92Y, K93R, P109S, I118T, N112S, andN177S, and conservative mutations thereof. In some embodiments, the oneor more amino acid substitution is A12T/H18L/M43V/F59L/E77K/P109S/I118T,K36G, K36G/L40M, E88D/K89R/D90K/A91G/F92Y/K93R,K89R/D90K/A91G/F92Y/K93R, E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S, orK89R/D90K/A91G/F92Y/K93R/N122S/N177S. In some embodiments, the variantCD80 polypeptide exhibits increased affinity to the ectodomain of CD28and the ectodomain of PD-L1 compared to a wildtype or unmodified CD80polypeptide, such as comprising the sequence set forth in SEQ ID NO: 28,152, or 372.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) corresponding to position(s) 4,29, 31, 36, 40, 41, 52, 67, 68, 70, 87, 88, 89, 90, 91, 92, 93, 107,109, 110, 118, 120, 130, 144, or 169 of SEQ ID NO: 28. In someembodiments, the variant CD80 polypeptide has one or more amino acidsubstitutions selected from the group consisting of V4M, R29H, Y31H,K36G, L40M, T41G, E52G, I67T, V68A, L70Q, Y87N, E88D, E88G, K89E, K89R,D90K, D90N, A91G, F92Y, K93R, D107N, P109S, T110A, I118V, T120S, T130A,N144D, and K169E. In some embodiments, the variant CD80 polypeptide hasone or more amino acid substitutions selected from the group consistingof V4M, R29H, Y31H, K36G, L40M, T41I, T41G, E52G, I67T, I69T, V68A,L70Q, Y87N, E88D, E88G, K89E, K89R, D90K, D90N, A91G, F92Y, K93R, D107N,P109S, T110A, I118V, T120S, T130A, N144D, and K169E and conservativeamino acid substitutions thereof. In some embodiments, the one or moreamino acid substitution is V4M/L70Q/A91G/T120S/T130A,V4M/L70Q/A91G/I118V/T120S/T130A/K169E,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,T41I/A91G, E52G/L70Q/A91G/T120S/T130A,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E, I67T/L70Q/A91G/T120S,I67T/L70Q/A91G/I118V/T120S, V68A/T110A, L70Q/A91G, L70Q/A91G/N144D,L70Q/A91G/T120S/T130A, L70Q/A91G/I118V/T120S/T130A/K169E,L70Q/A91G/T130A, K89R/D90K/A91G/F92Y/K93R,E88D/K89R/D90K/A91G/F92Y/K93R, A91G/I118V/T120S/T130A, A91G/T120S/T130Aor I69T/L70Q/A91G/T120S. In some embodiments, the variant CD80polypeptide exhibits increased affinity and/or increased selectivity tothe ectodomain of CTLA-4 compared to a wildtype or unmodified CD80polypeptide, such as comprising the sequence set forth in SEQ ID NO: 28,152, or 372. I In some embodiments, the variant CD80 polypeptide has oneor more amino acid modifications (e.g., substitutions) corresponding toposition(s) 36, 40, 52, 70, 88, 89, 90, 91, 92, 93, 107, 118120, 130,144, or 169 of SEQ ID NO: 28. In some embodiments, the variant CD80polypeptide has one or more amino acid substitutions selected from thegroup consisting of K36G, L40M, E52G, L70Q, E88D, K89R, D90K, A91G,F92Y, K93R, D107N, I118V, T120S, T130A, N144D, and K169E. In someembodiments, the variant CD80 polypeptide has one or more amino acidsubstitutions selected from the group consisting of K36G, L40M, E52G,L70Q, E88D, K89R, D90K, A91G, F92Y, K93R, D107N, I118V, T120S, T130AN144D, and K169E, and conservative amino acid substitutions thereof. Insome embodiments, the one or more amino acid substitution is K36G,K36G/L40M, E52G/L70Q/A91G/T120S/T130A,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E, L70Q/A91G,L70Q/A91G/N144D, L70Q/A91G/T120S/T130A,L70Q/A91G/I118V/T120S/T130A/K169E, E88D/K89R/D90K/A91G/F92Y/K93R, orK89R/D90K/A91G/F92Y/K93R. In some embodiments, the variant CD80polypeptide exhibits increased affinity for the ectodomain of CD28 andthe ectodomain of CTLA-4 compared to a wildtype or unmodified CD80polypeptide, such as comprising the sequence set forth in SEQ ID NO: 28,152, or 372.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) corresponding to position(s)29, 31, 36, 40, 41, 67, 70, 87, 88, 89, 90, 91, 92, 93, 109, 118, 120,122, 130, or 178 of SEQ ID NO: 28. In some embodiments, the variant CD80polypeptide has one or more amino acid substitutions selected from thegroup consisting of R29H, Y31H, K36G, L40M, T41G, I67T, L70Q, Y87N,E88D, E88G, K89E, K89R, D90N, D90K, A91G, F92Y, K93R, P109S, I118V,T120S. In some embodiments, the variant CD80 polypeptide has one or moreamino acid substitutions selected from the group consisting of R29H,Y31H, K36G, L40M, T41G, I67T, L70Q, Y87N, E88D, E88G, K89E, K89R, D90N,D90K, A91G, F92Y, K93R, P109S, I118V, T120S, and conservative amino acidsubstitutions thereof. In some embodiments, the one or more amino acidsubstitutions is R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G,K36G/L40M, I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R, or K89R/D90K/A91G/F92Y/K93R. In someembodiments, the variant CD80 polypeptide exhibits increased affinityfor the ectodomain of PD-L1 and the ectodomain of CTLA-4 compared towild-type or an unmodified CD80 polypeptide, such as comprising thesequence set forth in SEQ ID NO: 28, 152, or 372.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) corresponding to position(s)36, 40, 88 89, 90, 91, 92, or 93 of SEQ ID NO: 28. In some embodiments,the variant CD80 polypeptide has one or more amino acid substitutionsselected from the group consisting of K36G, L40M, E88D, K89R, D90K,A91G, F92Y, and K93R. In some embodiments, the variant CD80 polypeptidehas one or more amino acid substitutions selected from the groupconsisting of K36G, L40M, E88D, K89R, D90K, A91G, F92Y, K93R, andconservative amino acid substitutions thereof. In some embodiments, theone or more amino acid substitutions is K36G, K36G/L40M,E88D/K89R/D90K/A91G/F92Y/K93R, or K89R/D90K/A91G/F92Y/K93R. In someembodiments, the variant CD80 polypeptide exhibits increased affinityfor the ectodomain of CD28, the ectodomain of PD-L1 and the ectodomainof CTLA-4 compared to wild-type or an unmodified CD80 polypeptide, suchas comprising the sequence set forth in SEQ ID NO: 28, 152, or 372.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) corresponding to position(s)12, 18, 29, 31, 33, 36, 38, 40, 41, 42, 43, 47, 48, 63, 59, 64, 67, 68,70, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 97, 104, 109, 114, 115, 117,118, 120, 122, 126, 127, 130, 133, 140, 144, 148, 149, 168, 169, 177,183, 188 or 193 of SEQ ID NO: 28. In some embodiments, the variant CD80polypeptide has one or more amino acid substitutions selected from thegroup consisting of A12G, A12T, H18L, S21P, V22A, T28A, R29D, R29H,Y31H, Y31L, Q33H, K36G, M38I, LOM, T41A, T41G, M42T, M43R, M43V, M47T,N48I, F59L, N63S, N64S, I67T, V68A, V68M, L70Q, E77K, E81A, E81V, L85R,Y87N, E88D, E88G, K89E, K89N, D90K, D90N, A91G, A91T, F92P, F92Y, K93R,K93V, R94F, R94L, L97R, S103L, S103P, V104L, P109H, P109S, D115G, E117V,I118T, S114T, I118V, T120S, N122S, I126L, I127T, T130A, G133D, S140T,N144S, L148S, N149S, S168G, K169I, K169S, N177S, L183H, H188Q, R190S andQ193L. In some embodiments, the variant CD80 polypeptide has one or moreamino acid substitutions selected from the group consisting of R29D,R29H, Y31H, Y31L, Q33H, K36G, M38I, T41A, T41G, M42T, M43R, M47T, N63S,I67T, L70Q, E81A, E81V, L85R, Y87N, E88G, K89E, K89N, D90N, A91G, A91T,F92P, K93V, R94L, P109S, S114T, I118V, I118T, T120S, I127T, T130A,N144S, L148S, and N149S, and conservative amino acid substitutionsthereof. In some embodiments, the one or more amino acid substitutionsis N63S/L70Q/A91G/T120S/T130A, N63S/L70Q/A91G/S114T/I118V/T120S/T130A,L70Q/Y87N/A91G/T120S/I127T/T130A. In some embodiments, the one or moreamino acid substitutions is A12T/H18L/M43V/F59L/E77K/P109S/I118T,R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/I41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R, K89R/D90K/A91G/F92Y/K93R, A91G,H18L/R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/R190S,R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/E117V/I118T/N149S/S168G/H188Q,V22A/R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,R29D/Y31L/Q33H/K36G/M38I/I41A/M43R/M47T/N64S/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118T/T130A/N149S/K169I,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/D115G/I118T/T130A/G133D/N149S,T28A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/V104L/T130A/N149S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/N149S/H188Q,K89E/T130A, K89E/K93E/T130A,S21P/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N48I/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/S21P/N48I/V68A/P109H/I126L/K169I,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S,A12G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/T130A/L183H,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I/Q193L,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I,orR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/T130A/N149S/K169I.In some embodiments, the variant CD80 polypeptide exhibits increasedaffinity for the ectodomain of CD28 or the ectodomain of PD-L1, anddecreased affinity for the ectodomain of CTLA-4, compared to wild-typeor unmodified CD80 polypeptide, such as comprising the sequence setforth in SEQ ID NO: 28, 152, or 372.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) corresponding to position(s)63, 70, 81, 87, 91, 114, 118, 120, 127, or 130 of SEQ ID NO: 28. In someembodiments, the variant CD80 polypeptide has one or more amino acidsubstitutions selected from the group consisting of N63S, L70Q, E81A,Y87N, A91G, S114T, I118V, T120S, I127T, and T130A. In some embodiments,the variant CD80 polypeptide has one or more amino acid substitutionsselected from the group consisting of N63S, L70Q, E81A, Y87N, A91G,S114T, I118V, T120S, I127T, and T130A, and conservative amino acidsubstitutions thereof. In some embodiments, the one or more amino acidsubstitution isR29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S,N63S/L70Q/A91G/T120S/T130A, N63S/L70Q/A91G/S114T/I118V/T120S/T130A,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S, orL70Q/Y87N/A91G/T120S/I127T/T130A. In some embodiments, the variant CD80polypeptide exhibits increased affinity for the ectodomain of CD28, anddecreased affinity for the ectodomain of CTLA-4, compared to wild-typeor unmodified CD80 polypeptide, such as comprising the sequence setforth in SEQ ID NO: 28, 152, or 372.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) corresponding to position(s)12, 18, 29, 31, 33, 36, 38, 40, 41, 42, 43, 47, 48, 59, 64, 67, 68, 70,77, 81, 85, 87, 88, 89, 90, 91, 92, 93, 94, 97, 104, 109, 115, 117, 118,120, 122, 126, 130, 133, 140, 144, 148, 149, 168, 169, 177, 183, 188 or193 of SEQ ID NO: 28. In some embodiments, the variant CD80 polypeptidehas one or more amino acid substitutions selected from the groupconsisting of A12G, A12T, H18L, S21P, V22A, T28A, R29D, R29H, Y31H,Y31L, Q33H, K36G, M38I, L40M, T41A, T41G, M42T, M43R, M47T, M43V, N48I,F59L, N64S, I67T, V68A, V68M, L70Q, E77K, E81V, L85R, Y87N, E88D, E88G,K89E, K89N, K89R, D90K, D90N, A91G, A91T, F92P, F92Y, K93V, R94F, R94L,L97R, S103L, S103P, V104L, P109H, P109S, D115G, E117V, I118T, I118V,T120S, N122S, I126L, T130A, G133D, S140T, N144S, L148S, N149S, S168G,K169I, K169S, N177S, L183H, H188Q, R190S and Q193L. In some embodiments,the variant CD80 polypeptide has one or more amino acid substitutionsselected from the group consisting of R29D, R29H, Y31H, Y31L, Q33H,K36G, M38I, T41A, T41G, M42T, M43R, M47T, I67T, L70Q, E81V, L85R, Y87N,E88G, K89E, K89N, D90N, A91G, A91T, F92P, K93V, R94L, P109S, I118T,I118V, T120S, N144S, L148S, and N149S, and conservative amino acidsubstitutions thereof. In some embodiments, the one or more amino acidsubstitution isR29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S,I67T/L70Q/A91G/I118V/T120S, or I67T/L70Q/A91G/T120S. In someembodiments, the one or more amino acid substitutions isA12T/H18L/M43V/F59L/E77K/P109S/I118T,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R, K89R/D90K/A91G/F92Y/K93R, A91G,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/R190S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/E117V/I118T/N149S/S168G/H188Q,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N64S/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118T/T130A/N149S/K169I,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/D115G/I118T/T130A/G133D/N149S,T28A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/V104L/T130A/N149S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/N149S/H188Q,K89E/T130A, K89E/K93E/T130A,S21P/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N48I/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/S21P/N48I/V68A/P109H/I126L/K169I,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S,A12G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/T130A/L183H,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I/Q193L,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I,orR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/T130A/N149S/K169I.In some embodiments, the variant CD80 polypeptide exhibits increasedaffinity for the ectodomain of PD-L1, and decreased affinity for theectodomain of CTLA-4, compared to wild-type or unmodified CD80polypeptide, such as comprising the sequence set forth in SEQ ID NO: 28,152, or 372.

In some embodiments, the variant CD80 polypeptide has one or more aminoacid modifications (e.g., substitutions) corresponding to position(s)70, 81, 87, 91, or 120 of SEQ ID NO: 28. In some embodiments, thevariant CD80 polypeptide has one or more amino acid substitutionsselected from the group consisting of L70Q, Y87N, A91G, and T120S. Insome embodiments, the variant CD80 polypeptide has one or more aminoacid substitutions selected from the group consisting of L70Q, Y87N,A91G, and T120S, and conservative amino acid substitutions thereof. Insome embodiments, the variant CD80 polypeptide exhibits increasedaffinity for the ectodomain of CD28 and the ectodomain of PD-L1, anddecreased affinity for the ectodomain of CTLA-4, compared to wild-typeor unmodified CD80 polypeptide, such as comprising the sequence setforth in SEQ ID NO: 28, 152, or 372.

III. FORMAT OF VARIANT POLYPEPTIDES

The immunomodulatory polypeptide comprising a variant CD80 providedherein in which is contained a vIgD can be formatted in a variety ofways, including as a soluble protein, fusion or conjugate, membranebound protein, secreted protein or for delivery in an infectious agent.In some embodiments, the particular format can be chosen for the desiredtherapeutic application. In some cases, an immunomodulatory polypeptidecomprising a variant CD80 polypeptide is provided in a format toantagonize or block activity of its cognate binding partner, e.g. CD28,PD-L1 and/or CTLA-4. In some embodiments, antagonism of CTLA-4 may beuseful to promote immunity in oncology. In some embodiments, antagonismof CD28 may be useful for treating inflammation or autoimmunity. In somecases, an immunomodulatory polypeptide comprising a variant CD80polypeptide is provided in a format to agonize or stimulate activity ofits cognate binding partner, e.g. CD28, PD-L1 and/or CTLA-4. In someembodiments, agonism of CTLA-4 may be useful for treating inflammationor autoimmunity. In some embodiments, agonism of CD28 may be useful fortreating oncology. A skilled artisan can readily determine the activityof a particular format, such as for antagonizing or agonizing one ormore specific cognate binding partner. Exemplary methods for assessingsuch activities are provided herein, including in the examples.

In some aspects, provided are immunomodulatory proteins comprising avIgD of CD80 in which such proteins are soluble, e.g. fused to an Fcchain. In some aspects, one or more additional IgSF domain, such as oneor more additional vIgD, may be linked to a vIgD of CD80 as providedherein (hereinafter called a “stack” or “stacked” immunomodulatoryprotein). In some embodiments, the modular format of the providedimmunomodulatory proteins provides flexibility for engineering orgenerating immunomodulatory proteins for modulating activity of multiplecounterstructures (multiple cognate binding partners). In someembodiments, such “stack” molecules can be provided in a soluble formator, in some cases, may be provided as membrane bound or secretedproteins. In some embodiments, a variant CD80 immunomodulatory proteinis provided as a conjugate in which is contained a vIgD of CD80 linked,directly or indirectly, to a targeting agent or moiety, e.g. to anantibody or other binding molecules that specifically binds to a ligand,e.g. an antigen, for example, for targeting or localizing the vIgD to aspecific environment or cell, such as when administered to a subject. Insome embodiments, the targeting agent, e.g. antibody or other bindingmolecule, binds to a tumor antigen, thereby localizing the variant CD80containing the vIgD to the tumor microenvironment, for example, tomodulate activity of tumor infiltrating lymphocytes (TILs) specific tothe tumor microenvironment.

In some embodiments, provided immunomodulatory proteins are expressed incells and provided as part of an engineered cellular therapy (ECT). Insome embodiments, the variant CD80 polypeptide is expressed in a cell,such as an immune cell (e.g. T cell or antigen presenting cell), inmembrane-bound form, thereby providing a transmembrane immunomodulatoryprotein (hereinafter also called a “TIP”). In some embodiments,depending on the cognate binding partner recognized by the TIP,engineered cells expressing a TIP can agonize a cognate binding partnerby providing a costimulatory signal, either positive to negative, toother engineered cells and/or to endogenous T cells. In some aspects,the variant CD80 polypeptide is expressed in a cell, such as an immunecell (e.g. T cell or antigen presenting cell), in secretable form tothereby produce a secreted or soluble form of the variant CD80polypeptide (hereinafter also called a “SIP”), such as when the cellsare administered to a subject. In some aspects, a SIP can antagonize acognate binding partner in the environment (e.g. tumor microenvironment)in which it is secreted. In some embodiments, a variant CD80 polypeptideis expressed in an infectious agent (e.g. viral or bacterial agent)which, upon administration to a subject, is able to infect a cell invivo, such as an immune cell (e.g. T cell or antigen presenting cell),for delivery or expression of the variant polypeptide as a TIP or a SIPin the cell.

In some embodiments, a soluble immunomodulatory polypeptide, such as avariant CD80 containing a vIgD, can be encapsulated within a liposomewhich itself can be conjugated to any one of or any combination of theprovided conjugates (e.g., a targeting moiety). In some embodiments, thesoluble or membrane bound immunomodulatory polypeptides of the inventionare deglycosylated. In more specific embodiments, the variant CD80sequence is deglycosylated. In even more specific embodiments, the IgVand/or IgC (e.g. IgC2) domain or domains of the variant CD80 isdeglycosylated.

Non-limiting examples of provided formats are described in FIGS. 1A-1Cand further described below.

A. Soluble Protein

In some embodiments, the immunomodulatory protein containing a variantCD80 polypeptide is a soluble protein. Those of skill will appreciatethat cell surface proteins typically have an intracellular,transmembrane, and extracellular domain (ECD) and that a soluble form ofsuch proteins can be made using the extracellular domain or animmunologically active subsequence thereof. Thus, in some embodiments,the immunomodulatory protein containing a variant CD80 polypeptide lacksa transmembrane domain or a portion of the transmembrane domain. In someembodiments, the immunomodulatory protein containing a variant CD80lacks the intracellular (cytoplasmic) domain or a portion of theintracellular domain. In some embodiments, the immunomodulatory proteincontaining the variant CD80 polypeptide only contains the vIgD portioncontaining the ECD domain or a portion thereof containing an IgV domainand/or IgC (e.g. IgC2) domain or domains or specific binding fragmentsthereof containing the amino acid modification(s).

In some embodiments, an immunomodulatory polypeptide comprising avariant CD80 can include one or more variant CD80 polypeptides of theinvention. In some embodiments a polypeptide of the invention willcomprise exactly 1, 2, 3, 4, 5 variant CD80 sequences. In someembodiments, at least two of the variant CD80 sequences are identicalvariant CD80 sequences.

In some embodiments, the provided immunomodulatory polypeptide comprisestwo or more vIgD sequences of CD80. Multiple variant CD80 polypeptideswithin the polypeptide chain can be identical (i.e., the same species)to each other or be non-identical (i.e., different species) variant CD80sequences. In addition to single polypeptide chain embodiments, in someembodiments two, three, four, or more of the polypeptides of theinvention can be covalently or non-covalently attached to each other.Thus, monomeric, dimeric, and higher order (e.g., 3, 4, 5, or more)multimeric proteins are provided herein. For example, in someembodiments exactly two polypeptides of the invention can be covalentlyor non-covalently attached to each other to form a dimer. In someembodiments, attachment is made via interchain cysteine disulfide bonds.Compositions comprising two or more polypeptides of the invention can beof an identical species or substantially identical species ofpolypeptide (e.g, a homodimer) or of non-identical species ofpolypeptides (e.g., a heterodimer). A composition having a plurality oflinked polypeptides of the invention can, as noted above, have one ormore identical or non-identical variant CD80 polypeptides of theinvention in each polypeptide chain.

In some embodiments, the immunomodulatory protein comprises a variantCD80 polypeptide attached to an immunoglobulin Fc (yielding an“immunomodulatory Fc fusion,” such as a “variant CD80-Fc fusion,” alsotermed a CD80 vIgD-Fc fusion). In some embodiments, the attachment ofthe variant CD80 polypeptide is at the N-terminus of the Fc. In someembodiments, the attachment of the variant CD80 polypeptide is at theC-terminus of the Fc. In some embodiments, two or more CD80 variantpolypeptides (the same or different) are independently attached at theN-terminus and at the C-terminus.

In some embodiments, the Fc is murine or human Fc. In some embodiments,the Fc is a mammalian or human IgG1, IgG2, IgG3, or IgG4 Fc regions. Insome embodiments, the Fc is derived from IgG1, such as human IgG1. Insome embodiments, the Fc comprises the amino acid sequence set forth inSEQ ID NO: 226 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO: 226.

In some embodiments, the Fc region contains one more modifications toalter (e.g. reduce) one or more of its normal functions. In general, theFc region is responsible for effector functions, such ascomplement-dependent cytotoxicity (CDC) and antibody-dependent cellcytotoxicity (ADCC), in addition to the antigen-binding capacity, whichis the main function of immunoglobulins. Additionally, the FcRn sequencepresent in the Fc region plays the role of regulating the IgG level inserum by increasing the in vivo half-life by binding to an in vivo FcRnreceptor. In some embodiments, such functions can be reduced or alteredin an Fc for use with the provided Fc fusion proteins.

In some embodiments, one or more amino acid modifications may beintroduced into the Fc region of a CD80-Fc variant fusion providedherein, thereby generating an Fc region variant. In some embodiments,the Fc region variant has decreased effector function. There are manyexamples of changes or mutations to Fc sequences that can alter effectorfunction. For example, WO 00/42072, WO2006019447, WO2012125850,WO2015/107026, US2016/0017041 and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe exemplary Fc variants with improved ordiminished binding to FcRs. The contents of those publications arespecifically incorporated herein by reference.

In some embodiments, the provided variant CD80-Fc fusions comprise an Fcregion that exhibits reduced effector functions, which makes it adesirable candidate for applications in which the half-life of theCD80-Fc variant fusion in vivo is important yet certain effectorfunctions (such as CDC and ADCC) are unnecessary or deleterious. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theCD80-Fc variant fusion lacks FcγR binding (hence likely lacking ADCCactivity), but retains FcRn binding ability. The primary cells formediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays toassess ADCC activity of a molecule of interest is described in U.S. Pat.No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al.J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assaymethods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.; and CytoTox 96™ non-radioactive cytotoxicity assay(Promega, Madison, Wis.). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in an animal model such as thatdisclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).C1q binding assays may also be carried out to confirm that the CD80-Fcvariant fusion is unable to bind C1q and hence lacks CDC activity. See,e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. Toassess complement activation, a CDC assay may be performed (see, forexample, Gazzano-Santoro et al., J Immunol. Methods 202:163 (1996);Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivoclearance/half life determinations can also be performed using methodsknown in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol.18(12):1759-1769 (2006)).

CD80-Fc variant fusions with reduced effector function include thosewith substitution of one or more of Fc region residues 238, 265, 269,270, 297, 327 and 329 by EU numbering (U.S. Pat. No. 6,737,056). Such Fcmutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327 by EU numbering, including theso-called “DANA” Fc mutant with substitution of residues 265 and 297 toalanine (U.S. Pat. No. 7,332,581).

In some embodiments, the Fc region of CD80-Fv variant fusions has an Fcregion in which any one or more of amino acids at positions 234, 235,236, 237, 238, 239, 270, 297, 298, 325, and 329 (indicated by EUnumbering) are substituted with different amino acids compared to thenative Fc region. Such alterations of Fc region are not limited to theabove-described alterations, and include, for example, alterations suchas deglycosylated chains (N297A and N297Q), IgG1-N297G,IgG1-L234A/L235A, IgG1-L234A/L235E/G237A, IgG1-A325A/A330S/P331S,IgG1-C226S/C229S, IgG1-C226S/C229S/E233P/L234V/L235A,IgG1-E233P/L234V/L235A/G236del/S267K, IgG1-L234F/L235E/P331S,IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S,IgG4-L235A/G237A/E318A, and IgG4-L236E described in Current Opinion inBiotechnology (2009) 20 (6), 685-691; alterations such as G236R/L328R,L235G/G236R, N325A/L328R, and N325LL328R described in WO 2008/092117;amino acid insertions at positions 233, 234, 235, and 237 (indicated byEU numbering); and alterations at the sites described in WO 2000/042072.

Certain Fc variants with improved or diminished binding to FcRs aredescribed. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312,WO2006019447 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In some embodiments, there is provided a CD80-Fc variant fusioncomprising a variant Fc region comprising one or more amino acidsubstitutions which increase half-life and/or improve binding to theneonatal Fc receptor (FcRn). Antibodies with increased half-lives andimproved binding to FcRn are described in US2005/0014934A1 (Hinton etal.) or WO2015107026. Those antibodies comprise an Fc region with one ormore substitutions therein which improve binding of the Fc region toFcRn. Such Fc variants include those with substitutions at one or moreof Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 by EUnumbering, e.g., substitution of Fc region residue 434 (U.S. Pat. No.7,371,826).

In some embodiments, the Fc region of a CD80-Fc variant fusion comprisesone or more amino acid substitution E356D and M358L. In someembodiments, the Fc region of a CD80-Fc variant fusion comprises one ormore amino acid substitutions C220S, C226S, C229S. In some embodiments,the Fc region of a CD80 variant fusion comprises one or more amino acidsubstitutions R292C and V302C. See also Duncan & Winter, Nature322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351concerning other examples of Fc region variants.

In some embodiments, alterations are made in the Fc region that resultin diminished C1q binding and/or Complement Dependent Cytotoxicity(CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, andIdusogie et al., J. Immunol. 164: 4178-4184 (2000).

In some embodiments, there is provided a CD80-Fc variant fusioncomprising a variant Fc region comprising one or more amino acidmodifications, wherein the variant Fc region is derived from IgG1, suchas human IgG1. In some embodiments, the variant Fc region is derivedfrom the amino acid sequence set forth in SEQ ID NO: 226. In someembodiments, the Fc contains at least one amino acid substitution thatis N82G by numbering of SEQ ID NO: 226 (corresponding to N297G by EUnumbering). In some embodiments, the Fc further contains at least oneamino acid substitution that is R77C or V87C by numbering of SEQ ID NO:226 (corresponding to R292C or V302C by EU numbering). In someembodiments, the variant Fc region further comprises a C5S amino acidmodification by numbering of SEQ ID NO: 226 (corresponding to C220S byEU numbering). For example, in some embodiments, the variant Fc regioncomprises the following amino acid modifications: N82G and one or moreof the following amino acid modifications C5S, R77C or V87C withreference to SEQ ID NO:226.

In some embodiments, there is provided a CD80-Fc variant fusioncomprising a variant Fc region in which the variant Fc comprises thesequence of amino acids set forth in any of SEQ ID NOS:389, 392-394 and413, or a sequence of amino acids that exhibits at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to any of SEQ ID NOS: 389, 392-394 and 413.

In some embodiments, the Fc is derived from IgG2, such as human IgG2. Insome embodiments, the Fc comprises the amino acid sequence set forth inSEQ ID NO: 227 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO: 227.

In some embodiments, the Fc comprises the amino acid sequence set forthin SEQ ID NO: 411 or a sequence of amino acids that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity to SEQ ID NO: 411. In some embodiments,the IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 issubstituted with the CH3 domain of human IgG1 and which exhibitsinhibited aggregate formation, an antibody in which the CH3 and CH2domains of human IgG4 are substituted with the CH3 and CH2 domains ofhuman IgG1, respectively, or an antibody in which arginine at position409 indicated in the EU index proposed by Kabat et al. of human IgG4 issubstituted with lysine and which exhibits inhibited aggregate formation(see e.g. U.S. Pat. No. 8,911,726). In some embodiments, the Fc is anIgG4 containing the S228P mutation, which has been shown to preventrecombination between a therapeutic antibody and an endogenous IgG4 byFab-arm exchange (see e.g. Labrijin et al. (2009) Nat. Biotechnol.,27(8)767-71.) In some embodiments, the Fc comprises the amino acidsequence set forth in SEQ ID NO: 412 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 412.

In some embodiments, the variant CD80 polypeptide is directly linked tothe Fc sequence. In some embodiments, the variant CD80 polypeptide isindirectly linked to the Fc sequence, such as via a linker. In someembodiments, one or more “peptide linkers” link the variant CD80polypeptide and the Fc domain. In some embodiments, a peptide linker canbe a single amino acid residue or greater in length. In someembodiments, the peptide linker has at least one amino acid residue butis no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acid residues in length. In some embodiments, thelinker is (in one-letter amino acid code): GGGGS (“4GS”) or multimers ofthe 4GS linker, such as repeats of 2, 3, 4, or 5 4GS linkers.

In some embodiments, the variant CD80-Fc fusion protein is a dimerformed by two variant CD80 Fc polypeptides linked to an Fc domain. Insome specific embodiments, identical or substantially identical species(allowing for 3 or fewer N-terminus or C-terminus amino acid sequencedifferences) of CD80-Fc variant fusion polypeptides will be dimerized tocreate a homodimer. In some embodiments, the dimer is a homodimer inwhich the two variant CD80 Fc polypeptides are the same. Alternatively,different species of CD80-Fc variant fusion polypeptides can bedimerized to yield a heterodimer. Thus, in some embodiments, the dimeris a heterodimer in which the two variant CD80 Fc polypeptides aredifferent.

Also provided are nucleic acid molecules encoding the variant CD80-Fcfusion protein. In some embodiments, for production of an Fc fusionprotein, a nucleic acid molecule encoding a variant CD80-Fc fusionprotein is inserted into an appropriate expression vector. The resultingvariant CD80-Fc fusion protein can be expressed in host cellstransformed with the expression where assembly between Fc domains occursby interchain disulfide bonds formed between the Fc moieties to yielddimeric, such as divalent, variant CD80-Fc fusion proteins.

The resulting Fc fusion proteins can be easily purified by affinitychromatography over Protein A or Protein G columns. For the generationof heterodimers, additional steps for purification can be necessary. Forexample, where two nucleic acids encoding different variant CD80polypeptides are transformed into cells, the formation of heterodimersmust be biochemically achieved since variant CD80 molecules carrying theFc-domain will be expressed as disulfide-linked homodimers as well.Thus, homodimers can be reduced under conditions that favor thedisruption of interchain disulfides, but do no effect intra-chaindisulfides. In some cases, different variant-CD80 Fc monomers are mixedin equimolar amounts and oxidized to form a mixture of homo- andheterodimers. The components of this mixture are separated bychromatographic techniques. Alternatively, the formation of this type ofheterodimer can be biased by genetically engineering and expressing Fcfusion molecules that contain a variant CD80 polypeptide usingknob-into-hole methods described below.

B. Stack Molecules with Additional IgSF Domains

In some embodiments, the immunomodulatory proteins can contain any ofthe variant CD80 polypeptides provided herein linked, directly orindirectly, to one or more other immunoglobulin superfamily (IgSF)domain (“stacked” immunomodulatory protein construct and also called a“Type II” immunomodulatory protein). In some aspects, this can createunique multi-domain immunomodulatory proteins that bind two or more,such as three or more, cognate binding partners, thereby providing amulti-targeting modulation of the immune synapse.

In some embodiments, an immunomodulatory protein comprises a combination(a “non-wild-type combination”) and/or arrangement (a “non-wild typearrangement” or “non-wild-type permutation”) of a variant CD80 domainwith one or more other affinity modified and/or non-affinity modifiedIgSF domain sequences of another IgSF family member (e.g. a mammalianIgSF family member) that are not found in wild-type IgSF family members.In some embodiments, the immunomodulatory protein contains 2, 3, 4, 5 or6 immunoglobulin superfamily (IgSF) domains, where at least one of theIgSF domain is a variant CD80 IgSF domain (vIgD of CD80) according tothe provided description.

In some embodiments, the sequences of the additional IgSF domains can bea modified IgSF domain that contains one or more amino acidmodifications, e.g. substitutions, compared to a wildtype or unmodifiedIgSF domain. In some embodiments, the IgSF domain can be non-affinitymodified (e.g., wild-type) or have been affinity modified. In someembodiments, the unmodified or wild-type IgSF domain can be from mouse,rat, cynomolgus monkey, or human origin, or combinations thereof. Insome embodiments, the additional IgSF domains can be an IgSF domain ofan IgSF family member set forth in Table 2. In some embodiments, theadditional IgSF domain can be an affinity-modified IgSF domaincontaining one or more amino acid modifications, e.g. substitutions,compared to an IgSF domain contained in an IgSF family member set forthin Table 2.

In some embodiments, the additional IgSF domain is an affinity ornon-affinity modified IgSF domain contained in an IgSF family member ofa family selected from Signal-Regulatory Protein (SIRP) Family,Triggering Receptor Expressed On Myeloid Cells Like (TREML) Family,Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) Family,Sialic Acid Binding Ig-Like Lectin (SIGLEC) Family, Butyrophilin Family,B7 family, CD28 family, V-set and Immunoglobulin Domain Containing(VSIG) family, V-set transmembrane Domain (VSTM) family, MajorHistocompatibility Complex (MHC) family, Signaling lymphocyticactivation molecule (SLAM) family, Leukocyte immunoglobulin-likereceptor (LIR), Nectin (Nec) family, Nectin-like (NECL) family,Poliovirus receptor related (PVR) family, Natural cytotoxicitytriggering receptor (NCR) family, T cell immunoglobulin and mucin (TIM)family or Killer-cell immunoglobulin-like receptors (KIR) family. Insome embodiments, the additional IgSF domains are independently derivedfrom an IgSF protein selected from the group consisting of CD80(B7-1),CD86(B7-2), CD274 (PD-L1, B7-H1), PDCD1LG2(PD-L2, CD273), ICOSLG(B7RP1,CD275, ICOSL, B7-H2), CD276(B7-H3), VTCN1(B7-H4), CD28, CTLA-4,PDCD1(PD-1), ICOS, BTLA(CD272), CD4, CD8A(CD8-alpha), CD8B(CD8-beta),LAG3, HAVCR2(TIM-3), CEACAM1, TIGIT, PVR(CD155), PVRL2(CD112), CD226,CD2, CD160, CD200, CD200R1(CD200R), and NC R3 (NKp30).

The first column of Table 2 provides the name and, optionally, the nameof some possible synonyms for that particular IgSF member. The secondcolumn provides the protein identifier of the UniProtKB database, apublicly available database accessible via the internet at uniprot.orgor, in some cases, the GenBank Number. The Universal Protein Resource(UniProt) is a comprehensive resource for protein sequence andannotation data. The UniProt databases include the UniProt Knowledgebase(UniProtKB). UniProt is a collaboration between the EuropeanBioinformatics Institute (EMBL-EBI), the SIB Swiss Institute ofBioinformatics and the Protein Information Resource (PIR) and supportedmainly by a grant from the U.S. National Institutes of Health (NIH).GenBank is the NIH genetic sequence database, an annotated collection ofall publicly available DNA sequences (Nucleic Acids Research, 2013January; 41(D1):D36-42). The third column provides the region where theindicated IgSF domain is located. The region is specified as a rangewhere the domain is inclusive of the residues defining the range. Column3 also indicates the IgSF domain class for the specified IgSF region.Colum 4 provides the region where the indicated additional domains arelocated (signal peptide, S; extracellular domain, E; transmembranedomain, T; cytoplasmic domain, C). It is understood that description ofdomains can vary depending on the methods used to identify or classifythe domain, and may be identified differently from different sources.The description of residues corresponding to a domain in Table 2 is forexemplification only and can be several amino acids (such as one, two,three or four) longer or shorter. Column 5 indicates for some of thelisted IgSF members, some of its cognate cell surface binding partners.

TABLE 2 IgSF members according to the present disclosure. IgSF MemberAmino Acid Sequence Cognate Cell (SEQ ID NO) IgSF UniProtKB IgSF RegionSurface Precursor Member Protein & Domain Other Binding (mature(Synonym) Identifier Class Domains Partners residues) Mature ECD CD80NP_005182.1 35-135, 35- S: 1-34, CD28, CTLA-4, SEQ ID NO: 1 SEQ ID SEQID (B7-1) P33681 138, 37-138, or E: 35-242, PD-L1 (35-288) NO: 253 NO:28 35-141 IgV, T: 243-263, 145-230 or C: 264-288 154-232 IgC

CD86 P42081.2 33-131 IgV, S: 1-23, CD28, CTLA-4 SEQ ID NO: 2 SEQ ID SEQID (B7-2) 150-225 IgC2 E: 24-247, (24-329) NO: 254 NO: 29 T: 248-268, C:269-329 CD274 Q9NZQ7.1 24-130 IgV, S: 1-18, PD-1, B7-1 SEQ ID NO: 3 SEQID SEQ ID (PD-L1, 133-225 E: 19-238, (19-290) NO: 255 NO: 30 B7-H1) IgC2

T: 260-259, C: 260-290 PDCD1L Q9BQ51.2 21-118 IgV, S: 1-19, PD-1, RGMbSEQ ID NO: 4 SEQ ID SEQ ID G2 122-203 IgC2 E: 20-220, (20-273) NO: 256NO: 31 (PD-L2, T: 221-241, CD273) C: 242-273 ICOSLG O75144.2 19-129 IgV,ICOS, CD28, SEQ ID NO: 5 SEQ ID SEQ ID (B7RP1, 141-227 IgC2 S: 1-18,CTLA-4 (19-302) NO: 257 NO: 32 CD275, E: 19-256 ICOSL, T:257-277, B7-H2)C: 278-302 CD276 Q5ZPR3.1 29-139 IgV, S: 1-28, SEQ ID NO: 6 SEQ ID SEQID (B7-H3) 145-238 IgC2, E: 29-466, (29-534) NO: 258 NO: 33 243-357 IgV,T: 467-487, 367-453 IgC C: 488-534 VTCN1 Q7Z7D3.1 35-146 IgV, S: 1-24,SEQ ID NO: 7 SEQ ID SEQ ID (B7-H4) 153-241 IgV E: 25-259, (25-282) NO:259 NO: 34 T: 260-280, C: 281-282 CD28 P10747.1 28-137 IgV S: 1-18,B7-1, B7-2, SEQ ID NO: 8 SEQ ID SEQ ID E: 19-152, B7RP1 (19-220) NO: 260NO: 35 T: 153-179, C: 180-220 CTLA-4 P16410.3 39-140 IgV S: 1-35, B7-1,B7-2, SEQ ID NO: 9 SEQ ID SEQ ID E: 36-161, B7RP1 (36-223) NO: 261 NO:36 T: 162-182, C: 183-223 PDCD1 Q15116.3 35-145 IgV S:1-20, PD-L1, PD-L2SEQ ID NO: 10 SEQ ID SEQ ID (PD-1) E: 21-170, (21-288) NO: 262 NO: 37 T:171-191, C: 192-288 ICOS Q9Y6W8.1 30-132 IgV S: 1-20, B7RP1 SEQ ID NO:11 SEQ ID SEQ ID E: 21-140, (21-199) NO: 263 NO: 38 T: 141-161, C:162-199 BTLA Q7Z6A9.3 31-132 IgV S: 1-30, HVEM SEQ ID NO: 12 SEQ ID SEQID (CD272) E: 31-157, (31-289) NO: 264 NO: 39 T: 158-178, C: 179-289 CD4P01730.1 26-125 IgV, S: 1-25, MHC class II SEQ ID NO: 13 SEQ ID SEQ ID126-203 IgC2, E: 26-396, (26-458) NO: 265 NO: 40 204-317 IgC2, T:397-418, 317-389 IgC2 C: 419-458 CD8A P01732.1 22-135 IgV S: 1-21, E:MHC class I SEQ ID NO: 14 SEQ ID SEQ ID (CD8- 22-182, T: (22-235) NO:266 NO: 41 alpha) 183-203, C: 204-235 CD8B P10966.1 22-132 IgV S: 1-21,MHC class I SEQ ID NO: 15 SEQ ID SEQ ID (CD8- E: 22-170, (22-210) NO:267 NO: 42 beta) T: 171-191, C: 192-210 LAG3 P18627.5 37-167 IgV, S:1-28 MHC class II SEQ ID NO: 16 SEQ ID SEQ ID 168-252 IgC2, E: 29-450,(29-525) NO: 268 NO: 43 265-343 IgC2, T:451-471, 349-419 C: 472-525 IgC2

HAVCR2 Q8TDQ0.3 22-124 IgV S: 1-21, CEACAM-1, SEQ ID NO: 17 SEQ ID SEQID (TIM-3) E: 22-202, phosphatidyl- (22-301) NO: 269 NO: 44 T: 203-223,serine, C: 224-301 Galectin-9, HMGB1 CEACAM P13688.2 35-142 IgV, S:1-34, TIM-3 SEQ ID NO: 18 SEQ ID SEQ ID 145-232 IgC2, E: 35-428,(35-526) NO: 270 NO: 45 237-317 IgC2, T: 429-452, 323-413 IgC C: 453-526TIGIT Q495A1.1 22-124 IgV S: 1-21, CD155, CD112 SEQ ID NO: 19 SEQ ID SEQID E:22-141, (22-244) NO: 271 NO: 46 T: 142-162, C: 163-244 PVR P15151.224-139 IgV, S: 1-20 TIGIT, CD226, SEQ ID NO: 20 SEQ ID SEQ ID (CD155)145-237 IgC2, E: 21-343 CD96, (21-417) 244-328 T: 344-367, poliovirusNO: 272 NO: 47 IgC2

C: 368-417 PVRL2 Q92692.1 32-156 IgV, S: 1-31, TIGIT, CD226, SEQ ID NO:21 SEQ ID SEQ ID (CD112) 162-256 IgC2, E: 32-360, CD112R (32-538) NO:273 NO: 48 261-345 IgC2 T: 361-381, C: 382-538 CD226 Q15762.2 19-126IgC2, S: 1-18, CD155, CD112 SEQ ID NO: 22 SEQ ID SEQ ID 135-239 IgC2 E:19-254, (19-336) NO: 274 NO: 49 T: 255-275, C: 276-336 CD2 P06729.225-128 IgV, S: 1-24, CD58 SEQ ID NO: 23 SEQ ID SEQ ID 129-209 IgC2 E:25-209, (25-351) NO: 275 NO: 50 T: 210-235, C: 236-351 CD160 O95971.127-122 IgV S: 1-26 HVEM, MHC SEQ ID NO: 24 SEQ ID SEQ ID E: 27-122family of (27-159) NO: 276 NO: 51 proteins CD200 P41217.4 31-141 IgV, S:1-30, CD200R SEQ ID NO: 25 SEQ ID SEQ ID 142-232 IgC2 E: 31-232,(31-278) NO: 277 NO: 52 T: 233-259, C: 260-278 CD200R1 Q8TD46.2 53-139IgV, S: 1-28, CD200 SEQ ID NO: 26 SEQ ID SEQ ID (CD200R) 140-228 IgC2 E:29-243, (29-325) NO: 278 NO: 53 T: 244-264, C: 265-325 NCR3 O14931.119-126 IgC- S: 1-18, B7-H6 SEQ ID NO:27 SEQ ID SEQ ID (NKp30) like E:19-135, (19-201) NO: 279 NO: 54 T: 136-156, C: 157-201 VSIG8 Q5VU1322-141 IgV 1 S: 1-21 VISTA SEQ ID NO: 348 SEQ ID SEQ ID 146-257 E:22-263 (22-414) NO: 349 NO: 350 IgV 2 T: 264-284 C: 285-414

The number of such non-affinity modified or affinity modified IgSFdomains present in a “stacked” immunomodulatory protein construct(whether non-wild type combinations or non-wild type arrangements) is atleast 2, 3, 4, or 5 and in some embodiments exactly 2, 3, 4, or 5 IgSFdomains (whereby determination of the number of affinity modified IgSFdomains disregards any non-specific binding fractional sequences thereofand/or substantially immunologically inactive fractional sequencesthereof).

In some embodiments of a stacked immunomodulatory protein providedherein, the number of IgSF domains is at least 2 wherein the number ofaffinity modified and the number of non-affinity modified IgSF domainsis each independently at least: 0, 1, 2, 3, 4, 5, or 6. Thus, the numberof affinity modified IgSF domains and the number of non-affinitymodified IgSF domains, respectively, (affinity modified IgSF domain:non-affinity modified IgSF domain), can be exactly or at least: 2:0(affinity modified: wild-type), 0:2, 2:1, 1:2, 2:2, 2:3, 3:2, 2:4, 4:2,1:1, 1:3, 3:1, 1:4, 4:1, 1:5, or 5:1.

In some embodiments of a stacked immunomodulatory protein, at least twoof the non-affinity modified and/or affinity modified IgSF domains areidentical IgSF domains.

In some embodiments, a stacked immunomodulatory protein provided hereincomprises at least two affinity modified and/or non-affinity modifiedIgSF domains from a single IgSF member but in a non-wild-typearrangement (alternatively, “permutation”). One illustrative example ofa non-wild type arrangement or permutation is an immunomodulatoryprotein comprising a non-wild-type order of affinity modified and/ornon-affinity modified IgSF domain sequences relative to those found inthe wild-type CD80 whose IgSF domain sequences served as the source ofthe variant IgSF domains as provided herein. Thus, in one example, theimmunomodulatory protein can comprise an IgV proximal and an IgC distalto the transmembrane domain albeit in a non-affinity modified and/oraffinity modified form. The presence, in an immunomodulatory proteinprovided herein, of both non-wild-type combinations and non-wild-typearrangements of non-affinity modified and/or affinity modified IgSFdomains, is also within the scope of the provided subject matter.

In some embodiments of a stacked immunomodulatory protein, thenon-affinity modified and/or affinity modified IgSF domains arenon-identical (i.e., different) IgSF domains. Non-identical affinitymodified IgSF domains specifically bind, under specific bindingconditions, different cognate binding partners and are “non-identical”irrespective of whether or not the wild-type or unmodified IgSF domainsfrom which they are engineered was the same. Thus, for example, anon-wild-type combination of at least two non-identical IgSF domains inan immunomodulatory protein can comprise at least one IgSF domainsequence whose origin is from and unique to one CD80, and at least oneof a second IgSF domain sequence whose origin is from and unique toanother IgSF family member that is not CD80, wherein the IgSF domains ofthe immunomodulatory protein are in non-affinity modified and/oraffinity modified form. However, in alternative embodiments, the twonon-identical IgSF domains originate from the same IgSF domain sequencebut at least one is affinity modified such that they specifically bindto different cognate binding partners.

In some embodiments, the provided immunomodulatory proteins, in additionto containing a variant CD80 polypeptide, also contains at least 2, 3,4, 5 or 6 additional immunoglobulin superfamily (IgSF) domains, such asan IgD domain of an IgSF family member set forth in Table 2. In someembodiments, the provided immunomodulatory proteins contains at leastone additional IgSF domain (e.g. a second IgSF domain) in which at leastone additional or second IgSF domain is an IgSF domain set forth in awild-type or unmodified IgSF domain or a specific binding fragmentthereof contained in the sequence of amino acids set forth in any of SEQID NOS: 1-27 and 348. In some embodiments, the wild-type or unmodifiedIgSF domain is an IgV domain or an IgC domain, such as an IgC1 or IgC2domain.

In some embodiments, the provided immunomodulatory proteins, in additionto containing a variant CD80 polypeptide, also contains at least oneadditional IgSF domain (e.g. a second IgSF domain) that is a vIgD thatcontains one or more amino acid modifications (e.g. substitution,deletion or mutation) compared to an IgSF domain in a wild-type orunmodified IgSF domain, such as an IgSF domain in an IgSF family memberset forth in Table 2. In some embodiments, the additional or secondaffinity-modified IgSF domain comprises at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to a wild-type or unmodified IgSF domain or a specific bindingfragment thereof contained in the sequence of amino acids set forth inany of SEQ ID NOS: 1-27 and 348. In some embodiments, the wild-type orunmodified IgSF domain is an IgV domain or an IgC domain, such as anIgC1 or IgC2 domain. In some embodiments, the additional or second IgSFdomain is an affinity-modified IgV domain or IgC domain.

In some embodiments, the one or more additional IgSF domain (e.g. secondIgSF) domain is an IgSF domain (e.g. IgV) of another IgSF family memberthat binds or recognizes a tumor antigen. In such embodiments, the IgSFfamily member serves as a tumor-localizing moiety, thereby bringing thevIgD of CD80 in close proximity to immune cells in the tumormicroenvironment. In some embodiments, the additional IgSF domain (e.g.second IgSF) domain is an IgSF domain of NkP30, which binds orrecognizes B7-H6 expressed on a tumor cell. In some embodiments, the atleast one additional (e.g. second) IgSF domain, e.g. NkP30, is a vIgDthat contains one or more amino acid modifications (e.g. substitutions,deletions or additions). In some embodiments, the one or more amino acidmodifications increase binding affinity and/or selectivity to B7-H6compared to unmodified IgSF domain, e.g. NkP30, such as by at least orat least about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or50-fold. Among the exemplary polypeptides is an NKp30 variant thatcontains the mutations L30V/A60V/S64P/S86G with reference to positionsin the NKp30 extracellular domain corresponding to positions set forthin SEQ ID NO:54.

Tables 3-5 provide exemplary polypeptides containing one or moreaffinity-modified IgSF domains that can be used in stack constructsprovided herein.

TABLE 3 Exemplary variant ICOSL polypeptides ECD IgV SEQ SEQ Mutation(s)ID NO ID NO Wild-type 32 196 N52S 109 197 N52H 110 198 N52D 111 199N52Y/N57Y/F138L/L203P 112 200 N52H/N57Y/Q100P 113 201 N52S/Y146C/Y152C114 N52H/C198R 115 N52H/C140D/T225A 116 N52H/C198R/T225A 117 N52H/K92R118 202 N52H/S99G 119 203 N52Y 120 204 N57Y 121 205 N57Y/Q100P 122 206N52S/S130G/Y152C 123 N52S/Y152C 124 N52S/C198R 125 N52Y/N57Y/Y152C 126N52Y/N57Y/H129P/C198R 127 N52H/L161P/C198R 128 N52S/T113E 129 S54A 130207 N52D/S54P 131 208 N52K/L208P 132 209 N52S/Y152H 133 N52D/V151A 134N52H/I143T 135 N52S/L80P 136 210 F120S/Y152H/N201S 137 N52S/R75Q/L203P138 211 N52S/D158G 139 N52D/Q133H 140 N52S/N57Y/H94D/ 141 212L96F/L98F/Q100R N52S/N57Y/H94D/L96F/ 142 213 L98F/Q100R/G103E/F120SN52S/G103E 239 240 N52H/C140del/T225A 478

TABLE 4 Exemplary variant NKp30 polypeptides ECD IgC-like SEQ domain IDSEQ Mutation(s) NO ID NO Wild-type 54 214 L30V/A60V/ 143 215 S64P/S86GL30V 144 216 A60V 145 217 S64P 146 218 S86G 147 219

TABLE 5 Exemplary variant CD86 polypeptides ECD IgV ID SEQ SEQ IDMutation(s) NO NO Wild-type 29 220 Q35H/H90L/Q102H 148 221 Q35H 149 222H90L 150 223 Q102H 151 224

In some embodiments, the two or more IgSF domain, including a vIgD ofCD80 and one or more additional IgSF domain (e.g. second variant IgSFdomain) from another IgSF family member, are covalently ornon-covalently linked. A plurality of non-affinity modified and/oraffinity modified IgSF domains in a stacked immunomodulatory proteinpolypeptide chain need not be covalently linked directly to one another.In some embodiments, the two or more IgSF domains are linked directly orindirectly, such as via a linker. In some embodiments, an interveningspan of one or more amino acid residues indirectly covalently bonds IgSFdomains to each other. The linkage can be via the N-terminal toC-terminal residues. In some embodiments, the linkage can be made viaside chains of amino acid residues that are not located at theN-terminus or C-terminus of the IgSF domain(s). Thus, linkages can bemade via terminal or internal amino acid residues or combinationsthereof.

In some embodiments, one or more “peptide linkers” link the vIgD of CD80and an additional IgSF domain (e.g. second variant IgSF domain). In someembodiments, a peptide linker can be a single amino acid residue orgreater in length. In some embodiments, the peptide linker has at leastone amino acid residue but is no more than 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues inlength. In some embodiments, the linker is (in one-letter amino acidcode): GGGGS (“4GS”) or multimers of the 4GS linker, such as repeats of2, 3, 4, or 5 4GS linkers. In some embodiments, the peptide linker is(GGGGS)₂ or (GGGGS)₃. In some embodiments, the linker also can include aseries of alanine residues alone or in addition to another peptidelinker (such as a 4GS linker or multimer thereof). In some embodiments,the number of alanine residues in each series is: 2, 3, 4, 5, or 6alanines.

In some embodiments, the non-affinity modified and/or affinity modifiedIgSF domains are linked by “wild-type peptide linkers” inserted at theN-terminus and/or C-terminus of the first and/or second non-affinitymodified and/or affinity modified IgSF domains. These linkers are alsocalled leading sequences (N-terminal to non-affinity modified oraffinity modified IgSF domain) or trailing sequences (C-terminal tonon-affinity modified or affinity modified IgSF domain), and sequencesthat exist in the wild-type protein that span immediately outside thestructural prediction of the Ig fold of the IgSF. In some embodiments,the “wild-type linker” is an amino acid sequence that exists after thesignal sequence, but before in the IgSF domain, such as the defined IgVdomain, in the amino acid sequence of the wild-type protein. In someembodiments, the “wild-type” linker is an amino acid sequence thatexists immediately after the IgSF domain, such as immediately after thedefined IgV domain but before the IgC domain, in the amino acid sequenceof the wild-type protein. These linker sequences can contribute to theproper folding and function of the neighboring IgSF domain(s). In someembodiments, there is present a leading peptide linker inserted at theN-terminus of the first IgSF domain and/or a trailing sequence insertedat the C-terminus of the first non-affinity modified and/or affinitymodified IgSF domain. In some embodiments, there is present a secondleading peptide linker inserted at the N-terminus of the second IgSFdomain and/or a second trailing sequence inserted at the C-terminus ofthe second non-affinity modified and/or affinity modified IgSF domain.When the first and second non-affinity modified and/or affinity modifiedIgSF domains are derived from the same parental protein and areconnected in the same orientation, wild-type peptide linkers between thefirst and second non-affinity modified and/or affinity modified IgSFdomains are not duplicated. For example, when the first trailingwild-type peptide linker and the second leading wild-type peptide linkerare the same, the Type II immunomodulatory protein does not compriseeither the first trailing wild-type peptide linker or the second leadingwild-type peptide linker.

In some embodiments, the Type II immunomodulatory protein comprises afirst leading wild-type peptide linker inserted at the N-terminus of thefirst non-affinity modified and/or affinity modified IgSF domain,wherein the first leading wild-type peptide linker comprises at least 5(such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore) consecutive amino acids from the intervening sequence in thewild-type protein from which the first non-affinity modified and/oraffinity modified IgSF domain is derived between the parental IgSFdomain and the immediately preceding domain (such as a signal peptide oran IgSF domain). In some embodiments, the first leading wild-typepeptide linker comprises the entire intervening sequence in thewild-type protein from which the first non-affinity modified and/oraffinity modified IgSF domain is derived between the parental IgSFdomain and the immediately preceding domain (such as a signal peptide oran IgSF domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a first trailing wild-type peptide linker inserted at theC-terminus of the first non-affinity modified and/or affinity modifiedIgSF domain, wherein the first trailing wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the first non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain). In some embodiments, the firsttrailing wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the first non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a second leading wild-type peptide linker inserted at theN-terminus of the second non-affinity modified and/or affinity modifiedIgSF domain, wherein the second leading wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately preceding domain (such as asignal peptide or an IgSF domain). In some embodiments, the secondleading wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately preceding domain (such as asignal peptide or an IgSF domain).

In some embodiments, the Type II immunomodulatory protein furthercomprises a second trailing wild-type peptide linker inserted at theC-terminus of the second non-affinity modified and/or affinity modifiedIgSF domain, wherein the second trailing wild-type peptide linkercomprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more) consecutive amino acids from the interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain). In some embodiments, the secondtrailing wild-type peptide linker comprises the entire interveningsequence in the wild-type protein from which the second non-affinitymodified and/or affinity modified IgSF domain is derived between theparental IgSF domain and the immediately following domain (such as anIgSF domain or a transmembrane domain).

Exemplary trailing sequences for at Type II protein containing a CD80IgSF domain, can contain the amino acid sequence set forth in SEQ ID NO:232. Exemplary trailing sequences for a Type II protein containing aCD80 IgSF domain are set forth in SEQ ID NOS: 231, 232 and 371.Exemplary trailing sequences for a Type II protein containing an ICOSLIgSF domain are set forth in SEQ ID NOS: 233 and 234. Exemplary leadingand trailing sequences for a Type II protein containing a CD86 IgSFdomain are set forth in SEQ ID NOS: 236-238. An exemplary trailingsequence for a Type II protein containing an NKp30 IgSF domain is setforth in SEQ ID NO:235.

In some embodiments, the two or more IgSF domain, including a vIgD ofCD80 and one or more additional IgSF domain (e.g. second variant IgSFdomain) from another IgSF family member, are linked or attached to an Fcto form a dimeric multi-domain stack immunomodulatory protein. In someembodiments, the variant CD80 polypeptide and second IgSF domain areindependently linked, directly or indirectly, to the N- or C-terminus ofan Fc subunit. In some embodiments, the variant CD80 polypeptide andsecond IgSF domain are linked, directly or indirectly, and one of thevariant CD80 or second IgSF domain is also linked, directly orindirectly, to the N- or C-terminus of an Fc subunit. In someembodiments, linkage to the Fc is via a peptide linker, e.g. a peptidelinker, such as described above. In some embodiments, linkage betweenthe variant CD80 and second IgSF domain is via a peptide linker, e.g. apeptide linker, such as described above. In some embodiments, the vIgDof CD80, the one or more additional IgSF domains, and the Fc domain canbe linked together in any of numerous configurations as depicted in FIG.4. Exemplary configurations are described in the Examples.

In some embodiments, the stacked immunomodulatory protein is a dimerformed by two stacked immunomodulatory Fc fusion polypeptides. Alsoprovided are nucleic acid molecules encoding any of the stackedimmunomodulatory proteins. In some embodiments, the dimeric multi-domainstack immunomodulatory protein can be produced in cells by expression,or in some cases co-expression, of stack immunomodulatory Fc fusionpolypeptides, such as described above in according with generatingdimeric Fc fusion proteins.

In some embodiments, the dimeric multi-domain stack immunomodulatoryprotein is divalent for each Fc subunit, monovalent for each subunit, ordivalent for one subunit and tetravalent for the other.

In some embodiments, the dimeric multi-domain stack immunomodulatoryprotein is a homodimeric multi-domain stack Fc protein. In someembodiments, the dimeric multi-domain stack immunomodulatory proteincomprises a first stack immunomodulatory Fc fusion polypeptide and asecond stack immunomodulatory Fc fusion polypeptide in which the firstand second polypeptide are the same. In some embodiments, the Fc portionof the polypeptide can be any Fc as described above.

In some embodiments, the multi-domain stack molecule is heterodimeric,comprising two different Fc polypeptides wherein at least one is an Fcpolypeptide containing at least one variant CD80 polypeptide and/or atleast one second IgSF domain (e.g. second variant IgSF domain). In someembodiments, the multi-domain stack molecule contains a first Fcpolypeptide containing a variant CD80 and a second IgSF domain and asecond Fc polypeptide containing the variant CD80 and the second IgSFdomain. In some embodiments, the multi-domain stack molecule contains afirst Fc polypeptide containing a variant CD80 polypeptide and a secondIgSF domain and a second Fc polypeptide that is not linked to either avariant CD80 polypeptide or second IgSF domain.

In some embodiments, the multi-domain stack molecule contains a first Fcpolypeptides containing 1, 2, 3, 4 or more variant CD80 polypeptides and1, 2, 3, 4 or more second IgSF domains, wherein the total number of IgSFdomains in the first stack Fc polypeptide is greater than 2, 3, 4, 5, 6or more. In one example of such an embodiment, the second stack Fcpolypeptide contains 1, 2, 3, 4 or more variant CD80 polypeptides and 1,2, 3, 4 or more second IgSF domains, wherein the total number of IgSFdomains in the first stack Fc polypeptide is greater than 2, 3, 4, 5, 6or more. In another example of such an embodiments, the second Fcpolypeptide is not linked to either a variant CD80 polypeptide or secondIgSF domain.

In some embodiments, the heterodimeric stack molecule contains a firststack immunomodulatory Fc fusion polypeptide and a second stackimmunomodulatory Fc fusion polypeptide in which the first and secondpolypeptide are different. In some embodiments, a heterodimeric stackmolecule contains a first Fc subunit containing a first variant CD80polypeptide and/or second IgSF domain (e.g. second variant IgSF domain)and a second Fc subunit containing the other of the first variant CD80polypeptide or the second IgSF domain. In some embodiments, theheterodimeric stack molecule contains a first stack immunomodulatory Fcfusion polypeptide and a second stack immunomodulatory Fc fusionpolypeptide in which the first and second polypeptide are different. Insome embodiments, a heterodimeric stack molecule contains a first Fcsubunit containing a first variant CD80 polypeptide and/or second IgSFdomain (e.g. second variant IgSF domain) and a second Fc subunitcontaining both the first variant CD80 polypeptide and second IgSFdomain (e.g. second variant IgSF domain) but in a different orientationor configuration from the first Fc subunit.

In some embodiments, the Fc domain of one or both of the first andsecond stacked immunomodulatory Fc fusion polypeptide comprises amodification (e.g. substitution) such that the interface of the Fcmolecule is modified to facilitate and/or promote heterodimerization. Insome embodiments, modifications include introduction of a protuberance(knob) into a first Fc polypeptide and a cavity (hole) into a second Fcpolypeptide such that the protuberance is positionable in the cavity topromote complexing of the first and second Fc-containing polypeptides.Amino acids targeted for replacement and/or modification to createprotuberances or cavities in a polypeptide are typically interface aminoacids that interact or contact with one or more amino acids in theinterface of a second polypeptide.

In some embodiments, a first polypeptide that is modified to containprotuberance (hole) amino acids include replacement of a native ororiginal amino acid with an amino acid that has at least one side chainwhich projects from the interface of the first polypeptide and istherefore positionable in a compensatory cavity (hole) in an adjacentinterface of a second polypeptide. Most often, the replacement aminoacid is one which has a larger side chain volume than the original aminoacid residue. One of skill in the art knows how to determine and/orassess the properties of amino acid residues to identify those that areideal replacement amino acids to create a protuberance. In someembodiments, the replacement residues for the formation of aprotuberance are naturally occurring amino acid residues and include,for example, arginine (R), phenylalanine (F), tyrosine (Y), ortyrptophan (W). In some examples, the original residue identified forreplacement is an amino acid residue that has a small side chain suchas, for example, alanine, asparagines, aspartic acid, glycine, serine,threonine, or valine.

In some embodiments, a second polypeptide that is modified to contain acavity (hole) is one that includes replacement of a native or originalamino acid with an amino acid that has at least one side chain that isrecessed from the interface of the second polypeptide and thus is ableto accommodate a corresponding protuberance from the interface of afirst polypeptide. Most often, the replacement amino acid is one whichhas a smaller side chain volume than the original amino acid residue.One of skill in the art knows how to determine and/or assess theproperties of amino acid residues to identify those that are idealreplacement residues for the formation of a cavity. Generally, thereplacement residues for the formation of a cavity are naturallyoccurring amino acids and include, for example, alanine (A), serine (S),threonine (T) and valine (V). In some examples, the original amino acididentified for replacement is an amino acid that has a large side chainsuch as, for example, tyrosine, arginine, phenylalanine, or tryptophan.

The CH3 interface of human IgG1, for example, involves sixteen residueson each domain located on four anti-parallel β-strands which buries 1090Å2 from each surface (see e.g., Deisenhofer et al. (1981) Biochemistry,20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgwayet al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168).Modifications of a CH3 domain to create protuberances or cavities aredescribed, for example, in U.S. Pat. No. 5,731,168; International PatentApplications WO98/50431 and WO 2005/063816; and Ridgway et al., (1996)Prot. Engin., 9: 617-621. In some examples, modifications of a CH3domain to create protuberances or cavities are typically targeted toresidues located on the two central anti-parallel β-strands. The aim isto minimize the risk that the protuberances which are created can beaccommodated by protruding into the surrounding solvent rather thanbeing accommodated by a compensatory cavity in the partner CH3 domain.

In some embodiments, the heterodimeric molecule contains a T366Wmutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407Vmutations in the CH3 domain of the “hole chain”. In some cases, anadditional interchain disulfide bridge between the CH3 domains can alsobe used (Merchant, A. M., et al., Nature Biotech. 16 (1998) 677-681)e.g. by introducing a Y349C mutation into the CH3 domain of the “knobs”or “hole” chain and a E356C mutation or a S354C mutation into the CH3domain of the other chain. In some embodiments, the heterodimericmolecule contains S354C, T366W mutations in one of the two CH3 domainsand Y349C, T366S, L368A, Y407V mutations in the other of the two CH3domains. In some embodiments, the heterodimeric molecule comprisesE356C, T366W mutations in one of the two CH3 domains and Y349C, T366S,L368A, Y407V mutations in the other of the two CH3 domains. In someembodiments, the heterodimeric molecule comprises Y349C, T366W mutationsin one of the two CH3 domains and E356C, T366S, L368A, Y407V mutationsin the other of the two CH3 domains. In some embodiments, theheterodimeric molecule comprises Y349C, T366W mutations in one of thetwo CH3 domains and S354C, T366S, L368A, Y407V mutations in the other ofthe two CH3 domains. Examples of other knobs-in-holes technologies areknown in the art, e.g. as described by EP 1 870 459 A1.

In some embodiments, the Fc subunits of the heterodimeric moleculeadditionally can contain one or more other Fc mutation, such as anydescribed above. In some embodiments, the heterodimer molecule containsan Fc subunit with a mutation that reduces effector function.

In some embodiments, an Fc variant containing CH3 protuberance/cavitymodifications can be joined to a stacked immunomodulatory polypeptideanywhere, but typically via its N- or C-terminus, to the N- orC-terminus of a first and/or second stacked immunomodulatorypolypeptide, such as to form a fusion polypeptide. The linkage can bedirect or indirect via a linker. Typically, a knob and hole molecule isgenerated by co-expression of a first stacked immunomodulatorypolypeptide linked to an Fc variant containing CH3 protuberancemodification(s) with a second stacked immunomodulatory polypeptidelinked to an Fc variant containing CH3 cavity modification(s).

C. Conjugates and Fusions of Variant Polypeptides and ImmunomodulatoryProteins

In some embodiments, the variant polypeptides provided herein, which areimmunomodulatory proteins comprising variants of an Ig domain of theIgSF family (vIgD), can be conjugated with or fused with a moiety, suchas an effector moiety, such as another protein, directly or indirectly,to form a conjugate (“IgSF conjugate”). In some embodiments, theattachment can be covalent or non-covalent, e.g., via abiotin-streptavidin non-covalent interaction. In some embodiments of aCD80-Fc variant fusion, any one or combination of any two or more of theforegoing conjugates can be attached to the Fc or to the variant CD80polypeptide or to both

In some embodiments, the moiety can be a targeting moiety, a smallmolecule drug (non-polypeptide drug of less than 500 daltons molarmass), a toxin, a cytostatic agent, a cytotoxic agent, animmunosuppressive agent, a radioactive agent suitable for diagnosticpurposes, a radioactive metal ion for therapeutic purposes, aprodrug-activating enzyme, an agent that increases biological half-life,or a diagnostic or detectable agent.

In some embodiments the effector moiety is a therapeutic agent, such asa cancer therapeutic agent, which is either cytotoxic, cytostatic orotherwise provides some therapeutic benefit. In some embodiments, theeffector moiety is a targeting moiety or agent, such as an agent thattargets a cell surface antigen, e.g., an antigen on the surface of atumor cell. In some embodiments, the effector moiety is a label, whichcan generate a detectable signal, either directly or indirectly. In someembodiments, the effector moiety is a toxin. In some embodiments, theeffector moiety is a protein, peptide, nucleic acid, small molecule ornanoparticle.

In some embodiments, 1, 2, 3, 4, 5 or more effector moieties, which canbe the same or different, are conjugated, linked or fused to the variantpolypeptide or protein to form an IgSF conjugate. In some embodiments,such effector moieties can be attached to the variant polypeptide orimmunomodulatory protein using various molecular biological or chemicalconjugation and linkage methods known in the art and described below. Insome embodiments, linkers such as peptide linkers, cleavable linkers,non-cleavable linkers or linkers that aid in the conjugation reaction,can be used to link or conjugate the effector moieties to the variantpolypeptide or immunomodulatory protein.

In some embodiments, the IgSF conjugate comprises the followingcomponents: (protein or polypeptide), (L)_(q) and (effector moiety)_(m),wherein the protein or polypeptide is any of the described variantpolypeptides or immunomodulatory proteins capable of binding one or morecognate counter structure ligands as described; L is a linker forlinking the protein or polypeptide to the moiety; m is at least 1; q is0 or more; and the resulting IgSF conjugate binds to the one or morecounter structure ligands. In particular embodiments, m is 1 to 4 and qis 0 to 8.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a targeting agent that binds to a cell surface molecule,for example, for targeted delivery of the variant polypeptide orimmunomodulatory protein to a specific cell. In some embodiments, thetargeting agent is a molecule(s) that has the ability to localize andbind to a molecule present on a normal cell/tissue and/or tumorcell/tumor in a subject. In other words, IgSF conjugates comprising atargeting agent can bind to a ligand (directly or indirectly), which ispresent on a cell, such as a tumor cell. The targeting agents of theinvention contemplated for use include antibodies, polypeptides,peptides, aptamers, other ligands, or any combination thereof, that canbind a component of a target cell or molecule.

In some embodiments, the targeting agent binds a tumor cell(s) or canbind in the vicinity of a tumor cell(s) (e.g., tumor vasculature ortumor microenvironment) following administration to the subject. Thetargeting agent may bind to a receptor or ligand on the surface of thecancer cell. In another aspect of the invention, a targeting agent isselected which is specific for a noncancerous cells or tissue. Forexample, a targeting agent can be specific for a molecule presentnormally on a particular cell or tissue. Furthermore, in someembodiments, the same molecule can be present on normal and cancercells. Various cellular components and molecules are known. For example,if a targeting agent is specific for EGFR, the resulting IgSF conjugatecan target cancer cells expressing EGFR as well as normal skin epidermalcells expressing EGFR. Therefore, in some embodiments, an IgSF conjugateof the invention can operate by two separate mechanisms (targetingcancer and non-cancer cells).

In various aspects of the invention disclosed herein an IgSF conjugateof the invention comprises a targeting agent which can bind/target acellular component, such as a tumor antigen, a bacterial antigen, aviral antigen, a mycoplasm antigen, a fungal antigen, a prion antigen,an antigen from a parasite. In some aspects, a cellular component,antigen or molecule can each be used to mean, a desired target for atargeting agent. For example, in various embodiments, a targeting agentis specific for or binds to a component, which includes but is notlimited to, epidermal growth factor receptor (EGFR, ErbB-1, HERO, ErbB-2(HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family; insulin-likegrowth factor receptor (IGFR) family, IGF-binding proteins (IGFBPs),IGFR ligand family; platelet derived growth factor receptor (PDGFR)family, PDGFR ligand family; fibroblast growth factor receptor (FGFR)family, FGFR ligand family, vascular endothelial growth factor receptor(VEGFR) family, VEGF family; HGF receptor family; TRK receptor family;ephrin (EPH) receptor family; AXL receptor family; leukocyte tyrosinekinase (LTK) receptor family; TIE receptor family, angiopoietin 1,2;receptor tyrosine kinase-like orphan receptor (ROR) receptor family,e.g. ROR1; CD171 (L1CAM); B7-H6 (NCR3LG1); PD-L1, tumor glycosylationantigen, e.g. sTn or Tn, such as sTn Ag of MUC1; LHR (LHCGR);phosphatidylserine, discoidin domain receptor (DDR) family; RET receptorfamily; KLG receptor family; RYK receptor family; MuSK receptor family;Transfonning growth factor-α (TGF-α) receptors, TGF-β; Cytokinereceptors, Class I (hematopoietin family) and Class II (interferon/IL-10family) receptors, tumor necrosis factor (TNF) receptor superfamily(TNFRSF), death receptor family; cancer-testis (CT) antigens,lineage-specific antigens, differentiation antigens, alpha-actinin-4,ARTCl, breakpoint cluster region-Abelson (Bcr-abl) fusion products,B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-catenin (CTNNBl), celldivision cycle 27 (CDCl27), cyclin-dependent kinase 4 (CDK4), CDKN2A,COA-I, dek-can fusion protein, EFTUD-2, Elongation factor 2 (ELF2), Etsvariant gene6/acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusionprotein, fibronectin (FN), e.g. the extradomain A (EDA) of fibronectin,GPNMB, low density lipid receptor/GDP-L fucose: β-Dgalactose2-α-Lfucosyltransferase (LDLR/FUT) fusion protein, HLA-A2. arginine toisoleucine exchange at residue 170 of the α-helix of the α2-domain inthe HLA-A2gene (HLA-A*201-R170D, HLA-Al 1, heat shock protein 70-2mutated (HSP70-2M), K1AA0205, MART2, melanoma ubiquitous mutated 1, 2, 3(MUM-I, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, Myosin classI, NFYC, OGT, OS-9, pml-RARalpha fusion protein, PRDX5, PTPRK, K-ras(KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2fusion protein, Triosephosphate Isomerase, BAGE, BAGK-1, BAGE-2,3,4,5,GAGE-1,2,3,4,5,6,7,8, GnT-V (aberrant N-acetyl glucosaminyl transferaseV, MGAT5), HERV-K-MEL, KK-LC, KM-LAGE, LAGE-I, CTL-recognized antigen onmelanoma (CAMEL), MAGE-A1 (MAGE-I), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-AI 1, MAGE-A12, MAGE-3,MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, mucin 1 (MUC1),MART-1/Melan-A (MLANA), gp100, gp100/Pmel17 (SILV), tyrosinase (TYR),TRP-I, HAGE, NA-88, NY-ESO-I, NY-ESO-1/LAGE-2, SAGE, Spl7, SSX-1,2,3,4,TRP2-INT2, carcino-embryonic antigen (CEA), Kallikrein 4, mammaglobin-A,OAl, prostate specific antigen (PSA), TRP-1/gp75, TRP-2, adipophilin,interferon inducible protein absent in melanoma 2 (AIM-2), BING-4, CPSF,cyclin Dl, epithelial cell adhesion molecule (Ep-CAM), EphA3, fibroblastgrowth factor-5 (FGF-5), glycoprotein 250 (gp250), EGFR (ERBB1),HER-2/neu (ERBB2), interleukin 13 receptor α2 chain (IL13Rα2), IL-6receptor, intestinal carboxyl esterase (iCE), alpha-feto protein (AFP),M-CSF, mdm-2, MUC1, p53 (TP53), PBF, PRAME, PSMA, RAGE-I, RNF43, RU2AS,SOX10, STEAP1, survivin (BIRC5), human telomerase reverse transcriptase(hTERT), telomerase, Wilms' tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE,ADAM2, PAGE-5, LIP1, CTAGE-I, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85,AF15ql4, HCA661, LDHC, MORC, SGY-I, SPOl 1, TPX1, NY-SAR-35, FTHL17,NXF2, TDRD1, TEX15, FATE, TPTE, immunoglobulin idiotypes, Bence-Jonesprotein, estrogen receptors (ER), androgen receptors (AR), CD40, CD30,CD20, CD 19, CD33, cancer antigen 72-4 (CA 72-4), cancer antigen 15-3(CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125),cancer antigen 19-9 (CA 19-9), β-human chorionic gonadotropin, β-2microglobulin, squamous cell carcinoma antigen, neuron-specific enolase,heat shock protein gp96, GM2, sargramostim, CTLA-4, 707 alanine proline(707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4),carcinoembryogenic antigen peptide-1 (CAP-I), calcium-activated chloridechannel-2 (CLCA2), cyclophilin B (Cyp-B), human signet ring tumor-2(HST-2), Human papilloma virus (HPV) proteins (HPV-E6, HPV-E7, major orminor capsid antigens, others), Epstein-Barr virus (EBV) proteins (EBVlatent membrane proteins—LMP1, LMP2; others), Hepatitis B or C virusproteins, and HIV proteins.

In some embodiments, an IgSF conjugate, through its targeting agent,will bind a cellular component of a tumor cell, tumor vasculature ortumor microenvironment, thereby promoting killing of targeted cells viamodulation of the immune response, (e.g., by activation ofco-stimulatory molecules or inhibition of negative regulatory moleculesof immune cell activation), inhibition of survival signals (e.g., growthfactor or cytokine or hormone receptor antagonists), activation of deathsignals, and/or immune-mediated cytotoxicity, such as through antibodydependent cellular cytotoxicity. Such IgSF conjugates can functionthrough several mechanisms to prevent, reduce or eliminate tumor cells,such as to facilitate delivery of conjugated effector moieties to thetumor target, such as through receptor-mediated endocytosis of the IgSFconjugate; or such conjugates can recruit, bind, and/or activate immunecells (e.g. NK cells, monocytes/macrophages, dendritic cells, T cells, Bcells). Moreover, in some instances one or more of the foregoingpathways may operate upon administration of one or more IgSF conjugatesof the invention.

In some embodiments, an IgSF conjugate, through its targeting agent,will be localized to, such as bind to, a cellular component of a tumorcell, tumor vasculature or tumor microenvironment, thereby modulatingcells of the immune response in the vicinity of the tumor. In someembodiments, the targeting agent facilitates delivery of the conjugatedIgSF (e.g. vIgD) to the tumor target, such as to interact with itscognate binding partner to alter signaling of immune cells (e.g. NKcells, monocytes/macrophages, dendritic cells, T cells, B cells) bearingthe cognate binding partner. In some embodiments, localized deliverymediates costimulatory signaling via CD28 or ICOS.

In some embodiments, the targeting agent is an immunoglobulin. As usedherein, the term “immunoglobulin” includes natural or artificial mono-or polyvalent antibodies including, but not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, single chain Fv (scFv);anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, e.g., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass ofimmunoglobulin molecule.

In some embodiments, an IgSF conjugate, through its antibody targetingmoiety, will bind a cellular component of a tumor cell, tumorvasculature or tumor microenvironment, thereby promoting apoptosis oftargeted cells via modulation of the immune response, (e.g., byactivation of co-stimulatory molecules or inhibition of negativeregulatory molecules of immune cell activation), inhibition of survivalsignals (e.g., growth factor or cytokine or hormone receptorantagonists), activation of death signals, and/or immune-mediatedcytotoxicity, such as through antibody dependent cellular cytotoxicity.Such IgSF conjugates can function through several mechanisms to prevent,reduce or eliminate tumor cells, such as to facilitate delivery ofconjugated effector moieties to the tumor target, such as throughreceptor-mediated endocytosis of the IgSF conjugate; or such conjugatescan recruit, bind, and/or activate immune cells (e.g. NK cells,monocytes/macrophages, dendritic cells, T cells, B cells).

In some embodiments, an IgSF conjugate, through its antibody targetingmoiety, will bind a cellular component of a tumor cell, tumorvasculature or tumor microenvironment, thereby modulating the immuneresponse (e.g., by activation of co-stimulatory molecules or inhibitionof negative regulatory molecules of immune cell activation). In someembodiments, such conjugates can recognize, bind, and/or modulate (e.g.inhibit or activate) immune cells (e.g. NK cells, monocytes/macrophages,dendritic cells, T cells, B cells).

Antibody targeting moieties of the invention include antibody fragmentsthat include, but are not limited to, Fab, Fab′ and F(ab′)₂, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a VL or VH domain.Antigen-binding antibody fragments, including single-chain antibodies,may comprise the variable region(s) alone or in combination with theentirety or a portion of the following: hinge region, CH1, CH2, and CH3domains. Also included in the invention are antigen-binding fragmentsalso comprising any combination of variable region(s) with a hingeregion, CH1, CH2, and CH3 domains. Also included in the invention are Fcfragments, antigen-Fc fusion proteins, and Fc-targeting moietyconjugates or fusion products (Fc-peptide, Fc-aptamer). The antibodytargeting moieties of the invention may be from any animal originincluding birds and mammals. In one aspect, the antibody targetingmoieties are human, murine (e.g., mouse and rat), donkey, sheep, rabbit,goat, guinea pig, camel, horse, or chicken. Further, such antibodies maybe humanized versions of animal antibodies. The antibody targetingmoieties of the invention may be monospecific, bispecific, trispecific,or of greater multispecificity.

In various embodiments, an antibody/targeting moiety recruits, binds,and/or activates immune cells (e.g. NK cells, monocytes/macrophages,dendritic cells) via interactions between Fc (in antibodies) and Fcreceptors (on immune cells) and via the conjugated variant polypeptidesor immunomodulatory proteins provided herein. In some embodiments, anantibody/targeting moiety recognizes or binds a tumor agent via andlocalizes to the tumor cell the conjugated variant polypeptides orimmunomodulatory proteins provided herein to facilitate modulation ofimmune cells in the vicinity of the tumor.

Examples of antibodies which can be incorporated into IgSF conjugatesinclude but are not limited to antibodies such as Cetuximab (IMC-C225;Erbitux®), Trastuzumab (Herceptin®), Rituximab (Rituxan®; MabThera®),Bevacizumab (Avastin®), Alemtuzumab (Campath®; Campath-1H®;Mabcampath®), Panitumumab (ABX-EGF; Vectibix®), Ranibizumab (Lucentis®),Ibritumomab, Ibritumomab tiuxetan, (Zevalin®), Tositumomab, Iodine I 131Tositumomab (BEXXAR®), Catumaxomab (Removab®), Gemtuzumab, Gemtuzumabozogamicine (Mylotarg®), Abatacept (CTLA-4-Ig; Orencia®), Belatacept(L104EA29YIg; LEA29Y; LEA), Ipilimumab (MDX-010; MDX-101), Tremelimumab(ticilimumab; CP-675,206), PRS-010, PRS-050, Aflibercept (VEGF Trap,AVE005), Volociximab (M200), F200, MORAb-009, SS1P (CAT-5001),Cixutumumab (IMC-A12), Matuzumab (EMD72000), Nimotuzumab (h-R3),Zalutumumab (HuMax-EGFR), Necitumumab IMC-11F8, mAb806/ch806, Sym004,mAb-425, Panorex @ (17-1A) (murine monoclonal antibody); Panorex @(17-1A) (chimeric murine monoclonal antibody); IDEC-Y2B8 (murine,anti-CD2O MAb); BEC2 (anti-idiotypic MAb, mimics the GD epitope) (withBCG); Oncolym (Lym-1 monoclonal antibody); SMART MI95 Ab, humanized 13′I LYM-I (Oncolym), Ovarex (B43.13, anti-idiotypic mouse MAb); MDX-210(humanized anti-HER-2 bispecific antibody); 3622W94 MAb that binds toEGP40 (17-1A) pancarcinoma antigen on adenocarcinomas; Anti-VEGF,Zenapax (SMART Anti-Tac (IL-2 receptor); SMART MI95 Ab, humanized Ab,humanized); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-447(humanized anti-EGF receptor bispecific antibody); NovoMAb-G2(pancarcinoma specific Ab); TNT (chimeric MAb to histone antigens); TNT(chimeric MAb to histone antigens); Gliomab-H (Monoclons—Humanized Abs);GNI-250 Mab; EMD-72000 (chimeric-EGF antagonist); LymphoCide (humanizedLL2 antibody); and MDX-260 bispecific, targets GD-2, ANA Ab, SMART ID10Ab, SMART ABL 364 Ab or ImmuRAIT-CEA. As illustrated by the forgoinglist, it is conventional to make antibodies to a particular targetepitope.

In some embodiments, the antibody targeting moiety is a full lengthantibody, or antigen-binding fragment thereof, containing an Fc domain.In some embodiments, the variant polypeptide or immunomodulatory proteinis conjugated to the Fc portion of the antibody targeting moiety, suchas by conjugation to the N-terminus of the Fc portion of the antibody.

In some embodiments, the vIgD is linked, directly or indirectly, to theN- or C-terminus of the light and/or heavy chain of the antibody. Insome embodiments, linkage can be via a peptide linker, such as anydescribed above. Various configurations can be constructed. FIG. 6A-6Cdepict exemplary configurations. In some embodiments, the antibodyconjugate can be produced by co-expression of the heavy and light chainof the antibody in a cell.

In one aspect of the invention, the targeting agent is an aptamermolecule. For example, in some embodiments, the aptamer is comprised ofnucleic acids that function as a targeting agent. In variousembodiments, an IgSF conjugate of the invention comprises an aptamerthat is specific for a molecule on a tumor cell, tumor vasculature,and/or a tumor microenvironment. In some embodiments, the aptamer itselfcan comprise a biologically active sequence, in addition to thetargeting module (sequence), wherein the biologically active sequencecan induce an immune response to the target cell. In other words, suchan aptamer molecule is a dual use agent. In some embodiments, an IgSFconjugate of the invention comprises conjugation of an aptamer to anantibody, wherein the aptamer and the antibody are specific for bindingto separate molecules on a tumor cell, tumor vasculature, tumormicroenvironment, and/or immune cells.

The term “aptamer” includes DNA, RNA or peptides that are selected basedon specific binding properties to a particular molecule. For example, anaptamer(s) can be selected for binding a particular gene or gene productin a tumor cell, tumor vasculature, tumor microenvironment, and/or animmune cell, as disclosed herein, where selection is made by methodsknown in the art and familiar to one of skill in the art.

In some aspects of the invention the targeting agent is a peptide. Forexample, the variant polypeptides or immunomodulatory proteins providedherein can be conjugated to a peptide which can bind with a component ofa cancer or tumor cells. Therefore, such IgSF conjugates of theinvention comprise peptide targeting agents which binds to a cellularcomponent of a tumor cell, tumor vasculature, and/or a component of atumor microenvironment. In some embodiments, targeting agent peptidescan be an antagonist or agonist of an integrin. Integrins, whichcomprise an alpha and a beta subunit, include numerous types well knownto a skilled artisan.

In one embodiment, the targeting agent is Vvβ3. Integrin Vvβ3 isexpressed on a variety of cells and has been shown to mediate severalbiologically relevant processes, including adhesion of osteoclasts tobone matrix, migration of vascular smooth muscle cells, andangiogenesis. Suitable targeting molecules for integrins include RGDpeptides or peptidomimetics as well as non-RGD peptides orpeptidomimetics (see, e.g., U.S. Pat. Nos. 5,767,071 and 5,780,426) forother integrins such as V4.βi (VLA-4), V4-P7 (see, e.g., U.S. Pat. No.6,365,619; Chang et al, Bioorganic & Medicinal Chem Lett, 12:159-163(2002); Lin et al., Bioorganic & Medicinal Chem Lett, 12:133-136(2002)), and the like.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a therapeutic agent. In some embodiments, thetherapeutic agent includes, for example, daunomycin, doxorubicin,methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother.21:183-187, 1986). In some embodiments, the therapeutic agent has anintracellular activity. In some embodiments, the IgSF conjugate isinternalized and the therapeutic agent is a cytotoxin that blocks theprotein synthesis of the cell, therein leading to cell death. In someembodiments, the therapeutic agent is a cytotoxin comprising apolypeptide having ribosome-inactivating activity including, forexample, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin,diphtheria toxin, restrictocin, Pseudomonas exotoxin A and variantsthereof. In some embodiments, where the therapeutic agent is a cytotoxincomprising a polypeptide having a ribosome-inactivating activity, theIgSF conjugate must be internalized upon binding to the target cell inorder for the protein to be cytotoxic to the cells.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a toxin. In some embodiments, the toxin includes, forexample, bacterial toxins such as diphtheria toxin, plant toxins such asricin, small molecule toxins such as geldanamycin (Mandler et al., J.Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic &Med. Chem. Letters 10:1025-1028 (2000); Mandler et al., BioconjugateChem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc.Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode etal., Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342(1993)). The toxins may exert their cytotoxic and cytostatic effects bymechanisms including tubulin binding, DNA binding, or topoisomeraseinhibition.

In some embodiments, there is provided an IgSF conjugate comprising avariant polypeptide or immunomodulatory protein provided hereinconjugated with a label, which can generate a detectable signal,indirectly or directly. These IgSF conjugates can be used for researchor diagnostic applications, such as for the in vivo detection of cancer.The label is preferably capable of producing, either directly orindirectly, a detectable signal. For example, the label may beradio-opaque or a radioisotope, such as 3H, 14C, 32P, 35S, 123I, 125I,131I; a fluorescent (fluorophore) or chemiluminescent (chromophore)compound, such as fluorescein isothiocyanate, rhodamine or luciferin; anenzyme, such as alkaline phosphatase,β-galactosidase or horseradishperoxidase; an imaging agent; or a metal ion. In some embodiments, thelabel is a radioactive atom for scintigraphic studies, for example 99Tcor 123I, or a spin label for nuclear magnetic resonance (NMR) imaging(also known as magnetic resonance imaging, MRI), such as zirconium-89,iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,oxygen-17, gadolinium, manganese or iron. Zirconium-89 may be complexedto various metal chelating agents and conjugated to antibodies, e.g.,for PET imaging (WO 2011/056983). In some embodiments, the IgSFconjugate is detectable indirectly. For example, a secondary antibodythat is specific for the IgSF conjugate and contains a detectable labelcan be used to detect the IgSF conjugate.

The IgSF conjugates may be prepared using any methods known in the art.See, e.g., WO 2009/067800, WO 2011/133886, and U.S. Patent ApplicationPublication No. 2014322129, incorporated by reference herein in theirentirety.

The variant polypeptides or immunomodulatory proteins of an IgSFconjugate may be “attached to” the effector moiety by any means by whichthe variant polypeptides or immunomodulatory proteins can be associatedwith, or linked to, the effector moiety. For example, the variantpolypeptides or immunomodulatory proteins of an IgSF conjugate may beattached to the effector moiety by chemical or recombinant means.Chemical means for preparing fusions or conjugates are known in the artand can be used to prepare the IgSF conjugate. The method used toconjugate the variant polypeptides or immunomodulatory proteins andeffector moiety must be capable of joining the variant polypeptides orimmunomodulatory proteins with the effector moiety without interferingwith the ability of the variant polypeptides or immunomodulatoryproteins to bind to their one or more counter structure ligands.

The variant polypeptides or immunomodulatory proteins of an IgSFconjugate may be linked indirectly to the effector moiety. For example,the variant polypeptides or immunomodulatory proteins of an IgSFconjugate may be directly linked to a liposome containing the effectormoiety of one of several types. The effector moiety(s) and/or thevariant polypeptides or immunomodulatory proteins may also be bound to asolid surface.

In some embodiments, the variant polypeptides or immunomodulatoryproteins of an IgSF conjugate and the effector moiety are both proteinsand can be conjugated using techniques well known in the art. There areseveral hundred crosslinkers available that can conjugate two proteins.(See for example “Chemistry of Protein Conjugation and Crosslinking,”1991, Shans Wong, CRC Press, Ann Arbor). The crosslinker is generallychosen based on the reactive functional groups available or inserted onthe variant polypeptides or immunomodulatory proteins and/or effectormoiety. In addition, if there are no reactive groups, a photoactivatiblecrosslinker can be used. In certain instances, it may be desirable toinclude a spacer between the variant polypeptides or immunomodulatoryproteins and the effector moiety. Crosslinking agents known to the artinclude the homobifunctional agents: glutaraldehyde, dimethyladipimidateand Bis(diazobenzidine) and the heterobifunctional agents: mMaleimidobenzoyl-N-Hydroxysuccinimide and Sulfo-mMaleimidobenzoyl-N-Hydroxysuccinimide.

In some embodiments, the variant polypeptides or immunomodulatoryproteins of an IgSF conjugate may be engineered with specific residuesfor chemical attachment of the effector moiety. Specific residues usedfor chemical attachment of molecule known to the art include lysine andcysteine. The crosslinker is chosen based on the reactive functionalgroups inserted on the variant polypeptides or immunomodulatoryproteins, and available on the effector moiety.

An IgSF conjugate may also be prepared using recombinant DNA techniques.In such a case a DNA sequence encoding the variant polypeptides orimmunomodulatory proteins is fused to a DNA sequence encoding theeffector moiety, resulting in a chimeric DNA molecule. The chimeric DNAsequence is transfected into a host cell that expresses the fusionprotein. The fusion protein can be recovered from the cell culture andpurified using techniques known in the art.

Examples of attaching an effector moiety, which is a label, to thevariant polypeptides or immunomodulatory proteins include the methodsdescribed in Hunter, et al., Nature 144:945 (1962); David, et al.,Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219(1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel andMeares, Radioimmunoimaging And Radioimmunotherapy, Elsevier, N.Y.(1983); and Colcher et al., “Use Of Monoclonal Antibodies AsRadiopharmaceuticals For The Localization Of Human Carcinoma XenograftsIn Athymic Mice”, Meth. Enzymol., 121:802-16 (1986).

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as 99Tc or 123I, 186Re, 188Re and 111In can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al., Biochem. Biophys.Res. Commun. 80:49-57 (1978)) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the variant polypeptides or immunomodulatory proteins anda cytotoxic agent may be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate(SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate(SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters(such as dimethyl adipimidate HCl), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as toluene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described in Vitetta etal., Science 238:1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene tnaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See, e.g., WO94/11026. The linker maybe a “cleavable linker” facilitating release of the cytotoxic drug inthe cell. For example, an acid-labile linker, peptidase-sensitivelinker, photolabile linker, dimethyl linker or disulfide-containinglinker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No.5,208,020) may be used.

The IgSF conjugates of the invention expressly contemplate, but are notlimited to, drug conjugates prepared with cross-linker reagents: BMPS,EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) whichare commercially available (e.g., from Pierce Biotechnology, Inc.,Rockford, Ill., U.S.A). See pages 467-498, 2003-2004 ApplicationsHandbook and Catalog.

D. Transmembrane and Secretable Immunomodulatory Proteins and EngineeredCells

Provided herein are engineered cells which express the immunomodulatoryvariant CD80 polypeptides (alternatively, “engineered cells”). In someembodiments, the expressed immunomodulatory variant CD80 polypeptide isa transmembrane proteins and is surface expressed. In some embodiments,the expressed immunomodulatory variant CD80 polypeptide is expressed andsecreted from the cell.

1. Transmembrane Immunomodulatory Proteins

In some embodiments, an immunomodulatory polypeptide comprising avariant CD80 can be a membrane bound protein. As described in moredetail below, the immunomodulatory polypeptide can be a transmembraneimmunomodulatory polypeptide comprising a variant CD80 in which iscontained: an ectodomain containing at least one affinity modified IgSFdomain (IgV or IgC), a transmembrane domain and, optionally, acytoplasmic domain. In some embodiments, the transmembraneimmunomodulatory protein can be expressed on the surface of an immunecell, such as a mammalian cell, including on the surface of a lymphocyte(e.g. T cell or NK cell) or antigen presenting cell. In someembodiments, the transmembrane immunomodulatory protein is expressed onthe surface of a mammalian T-cell, including such T-cells as: a T helpercell, a cytotoxic T-cell (alternatively, cytotoxic T lymphocyte or CTL),a natural killer T-cell, a regulatory T-cell, a memory T-cell, or agamma delta T-cell. In some embodiments, the mammalian cell is anantigen presenting cell (APC). Typically, but not exclusively, theectodomain (alternatively, “extracellular domain”) of comprises the oneor more amino acid variations (e.g. amino acid substitutions) of thevariant CD80 of the invention. Thus, for example, in some embodiments atransmembrane protein will comprise an ectodomain that comprises one ormore amino acid substitutions of a variant CD80 of the invention.

In some embodiments, the engineered cells express a variant CD80polypeptides are transmembrane immunomodulatory polypeptides (TIPs) thatcan be a membrane protein such as a transmembrane protein. In typicalembodiments, the ectodomain of a membrane protein comprises anextracellular domain or IgSF domain thereof of a variant CD80 providedherein in which is contained one or more amino acid substitutions in atleast one IgSF domain as described. The transmembrane immunomodulatoryproteins provided herein further contain a transmembrane domain linkedto the ectodomain. In some embodiments, the transmembrane domain resultsin an encoded protein for cell surface expression on a cell. In someembodiments, the transmembrane domain is linked directly to theectodomain. In some embodiments, the transmembrane domain is linkedindirectly to the ectodomain via one or more linkers or spacers. In someembodiments, the transmembrane domain contains predominantly hydrophobicamino acid residues, such as leucine and valine.

In some embodiments, a full length transmembrane anchor domain can beused to ensure that the TIPs will be expressed on the surface of theengineered cell, such as engineered T cell. Conveniently, this could befrom a particular native protein that is being affinity modified (e.g.CD80 or other native IgSF protein), and simply fused to the sequence ofthe first membrane proximal domain in a similar fashion as the nativeIgSF protein (e.g. CD80). In some embodiments, the transmembraneimmunomodulatory protein comprises a transmembrane domain of thecorresponding wild-type or unmodified IgSF member, such as atransmembrane domain contained in the sequence of amino acids set forthin SEQ ID NO:1 (Table 2). In some embodiments, the membrane bound formcomprises a transmembrane domain of the corresponding wild-type orunmodified polypeptide, such as corresponding to residues 243-263 of SEQID NO:1.

In some embodiments, the transmembrane domain is a non-nativetransmembrane domain that is not the transmembrane domain of nativeCD80. In some embodiments, the transmembrane domain is derived from atransmembrane domain from another non-CD80 family member polypeptidethat is a membrane-bound or is a transmembrane protein. In someembodiments, a transmembrane anchor domain from another protein on Tcells can be used. In some embodiments, the transmembrane domain isderived from CD8. In some embodiments, the transmembrane domain canfurther contain an extracellular portion of CD8 that serves as a spacerdomain. An exemplary CD8 derived transmembrane domain is set forth inSEQ ID NO: 246 or 399 or a portion thereof containing the CD8transmembrane domain. In some embodiments, the transmembrane domain is asynthetic transmembrane domain.

In some embodiments, the transmembrane immunomodulatory protein furthercontains an endodomain, such as a cytoplasmic signaling domain, linkedto the transmembrane domain. In some embodiments, the cytoplasmicsignaling domain induces cell signaling. In some embodiments, theendodomain of the transmembrane immunomodulatory protein comprises thecytoplasmic domain of the corresponding wild-type or unmodifiedpolypeptide, such as a cytoplasmic domain contained in the sequence ofamino acids set forth in SEQ ID NO:1 (see Table 2).

In some embodiments, a provided transmembrane immunomodulatory proteinthat is or comprises a variant CD80 comprises a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 253 andcontains an ectodomain comprising at least one affinity-modified CD80IgSF domain as described and a transmembrane domain. In someembodiments, the transmembrane immunomodulatory protein contains any oneor more amino acid substitutions in an IgSF domain (e.g. IgV domain) asdescribed, including any set forth in Table 1. In some embodiments, thetransmembrane immunomodulatory protein can further comprise acytoplasmic domain as described. In some embodiments, the transmembraneimmunomodulatory protein can further contain a signal peptide. In someembodiments, the signal peptide is the native signal peptide ofwild-type IgSF member, such as contained in the sequence of amino acidsset forth in SEQ ID NO:1 (see e.g. Table 2).

Also provided is a nucleic acid molecule encoding such transmembraneimmunomodulatory proteins. In some embodiments, a nucleic acid moleculeencoding a transmembrane immunomodulatory protein comprises a nucleotidesequence that encodes a sequence of amino acids that exhibits at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to SEQ ID NOS: 253 and contains an ectodomaincomprising at least one affinity-modified IgSF domain as described, atransmembrane domain and, optionally, a cytoplasmic domain. In someembodiments, the nucleic acid molecule can further comprise a sequenceof nucleotides encoding a signal peptide. In some embodiments, thesignal peptide is the native signal peptide of the correspondingwild-type IgSF member (see e.g. Table 2).

An example of a transmembrane immunomodulatory protein is a CD80 TIPcomprising i) the sequence of amino acids set forth in SEQ ID NO:241 orii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO:241 and that comprises the affinity-modifieddomain of SEQ ID NO:241 or the amino acid substitutions containedtherein. Also provided is i) a sequence of nucleotides set forth in SEQID NO:242, ii) a sequence that exhibits at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 242 and that encodes a TIP that comprises theaffinity-modified domain of SEQ ID NO:241 or a polypeptide that containsthe amino acid substitutions of SEQ ID NO:241, or iii) a sequence of i)or ii) having degenerate codons.

In some embodiments, provided are CAR-related transmembraneimmunomodulatory proteins in which the endodomain of a transmembraneimmunomodulatory protein comprises a cytoplasmic signaling domain thatcomprises at least one ITAM (immunoreceptor tyrosine-based activationmotif)-containing signaling domain. ITAM is a conserved motif found in anumber of protein signaling domains involved in signal transduction ofimmune cells, including in the CD3-zeta chain (“CD3-z”) involved inT-cell receptor signal transduction. In some embodiments, the endodomaincomprises at CD3-zeta signaling domain. In some embodiments, theCD3-zeta signaling domain comprises the sequence of amino acids setforth in SEQ ID NO: 333 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% to SEQ ID NO:247 and retains the activity of T cellsignaling. In some embodiments, the endodomain of a CAR-relatedtransmembrane immunomodulatory protein can further comprise acostimulatory signaling domain to further modulate immunomodulatoryresponses of the T-cell. In some embodiments, the costimulatorysignaling domain is CD28, ICOS, 41BB or OX40. In some embodiments, thecostimulatory signaling domain is a derived from CD28 or 4-1BB andcomprises the sequence of amino acids set forth in any of SEQ ID NOS:400-403 or a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQID NO:400-403 and retains the activity of T cell costimulatorysignaling. In some embodiments, the provided CAR-related transmembraneimmunomodulatory proteins have features of CARs to stimulate T cellsignaling upon binding of an affinity modified IgSF domain to a cognatebinding partner or counter structure. In some embodiments, upon specificbinding by the affinity-modified IgSF domain to its counter structurecan lead to changes in the immunological activity of the T-cell activityas reflected by changes in cytotoxicity, proliferation or cytokineproduction.

In some embodiments, the transmembrane immunomodulatory protein does notcontain an endodomain capable of mediating cytoplasmic signaling. Insome embodiments, the transmembrane immunomodulatory protein lacks thesignal transduction mechanism of the wild-type or unmodified polypeptideand therefore does not itself induce cell signaling. In someembodiments, the transmembrane immunomodulatory protein lacks anintracellular (cytoplasmic) domain or a portion of the intracellulardomain of the corresponding wild-type or unmodified polypeptide, such asa cytoplasmic signaling domain contained in the sequence of amino acidsset forth in SEQ ID NO:1 (see Table 2). In some embodiments, thetransmembrane immunomodulatory protein does not contain an ITIM(immunoreceptor tyrosine-based inhibition motif), such as contained incertain inhibitory receptors, including inhibitory receptors of the IgSFfamily (e.g. PD-1 or TIGIT). Thus, in some embodiments, thetransmembrane immunomodulatory protein only contains the ectodomain andthe transmembrane domain, such as any as described.

2. Secreted Immunomodulatory Proteins and Engineered Cells

In some embodiments, the CD80 variant immunomodulatory polypeptidecontaining any one or more of the amino acid mutations as describedherein, is secretable, such as when expressed from a cell. Such avariant CD80 immunomodulatory protein does not comprise a transmembranedomain. In some embodiments, the variant CD80 immunomodulatory proteinis not conjugated to a half-life extending moiety (such as an Fc domainor a multermization domain). In some embodiments, the variant CD80immunomodulatory protein comprises a signal peptide, e.g. an antibodysignal peptide or other efficient signal sequence to get domains outsideof cell. When the immunomodulatory protein comprises a signal peptideand is expressed by an engineered cell, the signal peptide causes theimmunomodulatory protein to be secreted by the engineered cell.Generally, the signal peptide, or a portion of the signal peptide, iscleaved from the immunomodulatory protein with secretion. Theimmunomodulatory protein can be encoded by a nucleic acid (which can bepart of an expression vector). In some embodiments, the immunomodulatoryprotein is expressed and secreted by a cell (such as an immune cell, forexample a primary immune cell).

Thus, in some embodiments, there are provided variant CD80immunomodulatory proteins that further comprises a signal peptide. Insome embodiments, provided herein is a nucleic acid molecule encodingthe variant CD80 immunomodulatory protein operably connected to asecretion sequence encoding the signal peptide.

A signal peptide is a sequence on the N-terminus of an immunomodulatoryprotein that signals secretion of the immunomodulatory protein from acell. In some embodiments, the signal peptide is about 5 to about 40amino acids in length (such as about 5 to about 7, about 7 to about 10,about 10 to about 15, about 15 to about 20, about 20 to about 25, orabout 25 to about 30, about 30 to about 35, or about 35 to about 40amino acids in length).

In some embodiments, the signal peptide is a native signal peptide fromthe corresponding wild-type CD80 (see Table 2). In some embodiments, thesignal peptide is a non-native signal peptide. For example, in someembodiments, the non-native signal peptide is a mutant native signalpeptide from the corresponding wild-type CD80, and can include one ormore (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitutionsinsertions or deletions. In some embodiments, the non-native signalpeptide is a signal peptide or mutant thereof of a family member fromthe same IgSF family as the wild-type IgSF family member. In someembodiments, the non-native signal peptide is a signal peptide or mutantthereof from an IgSF family member from a different IgSF family that thewild-type IgSF family member. In some embodiments, the signal peptide isa signal peptide or mutant thereof from a non-IgSF protein family, suchas a signal peptide from an immunoglobulin (such as IgG heavy chain orIgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), orCD33), a serum albumin protein (e.g. HSA or albumin), a human azurocidinpreprotein signal sequence, a luciferase, a trypsinogen (e.g.chumotrypsinogen or trypsinogen) or other signal peptide able toefficiently secrete a protein from a cell. Exemplary signal peptidesinclude any described in the Table 6.

TABLE 6 Exemplary Signal Peptides SEQ ID NO Signal PeptidePeptide Sequence SEQ ID NO: 353 HSA signal peptide MKWVTFISLLFLFSSAYSSEQ ID NO: 354 Ig kappa light chain MDMRAPAGIFGFLLVLFPGYRSSEQ ID NO: 355 human azurocidin preprotein signal MTRLTVLALLAGLLASSRAsequence SEQ ID NO: 356 IgG heavy chain signal peptideMELGLSWIFLLAILKGVQC SEQ ID NO: 357 IgG heavy chain signal peptideMELGLRWVFLVAILEGVQC SEQ ID NO: 358 IgG heavy chain signal peptideMKHLWFFLLLVAAPRWVLS SEQ ID NO: 359 IgG heavy chain signal peptideMDWTWRILFLVAAATGAHS SEQ ID NO: 360 IgG heavy chain signal peptideMDWTWRFLFVVAAATGVQS SEQ ID NO: 361 IgG heavy chain signal peptideMEFGLSWLFLVAILKGVQC SEQ ID NO: 362 IgG heavy chain signal peptideMEFGLSWVFLVALFRGVQC SEQ ID NO: 363 IgG heavy chain signal peptideMDLLHKNMKHLWFFLLLVAAPRWVLS SEQ ID NO: 364 IgG Kappa light chain signalMDMRVPAQLLGLLLLWLSGARC sequences: SEQ ID NO: 365IgG Kappa light chain signal MKYLLPTAAAGLLLLAAQPAMA sequences:SEQ ID NO: 366 Gaussia luciferase MGVKVLFALICIAVAEA SEQ ID NO: 367Human albumin MKWVTFISLLFLFSSAYS SEQ ID NO: 368 Human chymotrypsinogenMAFLWLLSCWALLGTTFG SEQ ID NO: 369 Human interleukin-2 MQLLSCIALILALVSEQ ID NO: 370 Human bypsinogen-2 MNLLLILTFVAAAVA

In some embodiments of a secretable variant CD80 immunomodulatoryprotein, the immunomodulatory protein comprises a signal peptide whenexpressed, and the signal peptide (or a portion thereof) is cleaved fromthe immunomodulatory protein upon secretion.

In some embodiments, the engineered cells express a variant CD80polypeptides that are secreted from the cell. In some embodiments, sucha variant CD80 polypeptide is encoded by a nucleic acid moleculeencoding an immunomodulatory protein under the operable control of asignal sequence for secretion. In some embodiments, the encodedimmunomodulatory protein is secreted when expressed from a cell. In someembodiments, the immunomodulatory protein encoded by the nucleic acidmolecule does not comprise a transmembrane domain. In some embodiments,the immunomodulatory protein encoded by the nucleic acid molecule doesnot comprise a half-life extending moiety (such as an Fc domain or amultimerization domain). In some embodiments, the immunomodulatoryprotein encoded by the nucleic acid molecule comprises a signal peptide.In some embodiments, a nucleic acid of the invention further comprisesnucleotide sequence that encodes a secretory or signal peptide operablylinked to the nucleic acid encoding the immunomodulatory protein,thereby allowing for secretion of the immunomodulatory protein

3. Cells and Engineering Cells

Provided herein are engineered cells expressing any of the providedimmunomodulatory polypeptide. In some embodiments, the engineered cellsexpress on their surface any of the provided transmembraneimmunomodulatory polypeptides. In some embodiments, the engineered cellsexpress and are capable of or are able to secrete the immunomodulatoryprotein from the cells under conditions suitable for secretion of theprotein. In some embodiments, the immunomodulatory protein is expressedon a lymphocyte such as a tumor infiltrating lymphocyte (TIL), T-cell orNK cell, or on a myeloid cell. In some embodiments, the engineered cellsare antigen presenting cells (APCs). In some embodiments, the engineeredcells are engineered mammalian T-cells or engineered mammalian antigenpresenting cells (APCs). In some embodiments, the engineered T-cells orAPCs are human or murine cells.

In some embodiments, engineered T-cells include, but are not limited to,T helper cell, cytotoxic T-cell (alternatively, cytotoxic T lymphocyteor CTL), natural killer T-cell, regulatory T-cell, memory T-cell, orgamma delta T-cell. In some embodiments, the engineered T cells are CD4+or CD8+. In addition to the signal of the MHC, engineered T-cells alsorequire a co-stimulatory signal, which in some embodiments is providedby a variant CD80 transmembrane immunomodulatory polypeptide expressedin membrane bound form as discussed previously.

In some embodiments, the engineered APCs include, for example, MHC IIexpressing APCs such as macrophages, B cells, and dendritic cells, aswell as artificial APCs (aAPCs) including both cellular and acellular(e.g., biodegradable polymeric microparticles) aAPCs. Artificial APCs(aAPCs) are synthetic versions of APCs that can act in a similar mannerto APCs in that they present antigens to T-cells as well as activatethem. Antigen presentation is performed by the MHC (Class I or ClassII). In some embodiments, in engineered APCs such as aAPCs, the antigenthat is loaded onto the MHC is, in some embodiments, a tumor specificantigen or a tumor associated antigen. The antigen loaded onto the MHCis recognized by a T-cell receptor (TCR) of a T cell, which, in somecases, can express CTLA-4 or CD80 or other molecule recognized by thevariant CD80 polypeptides provided herein. Materials which can be usedto engineer an apace include: poly (glycolic acid),poly(lactic-co-glycolic acid), iron-oxide, liposomes, lipid bilayers,sepharose, and polystyrene.

In some embodiments, an immunomodulatory protein, such as atransmembrane immunomodulatory protein or a secretable immunomodulatoryprotein, provided herein is co-expressed or engineered into a cell thatexpresses an antigen-binding receptor, such as a recombinant receptor,such as a chimeric antigen receptor (CAR) or T cell receptor (TCR). Insome embodiments, the engineered cell, such as an engineered T cell,recognizes a desired antigen associated with cancer, inflammatory andautoimmune disorders, or a viral infection. In specific embodiments, theantigen-binding receptor contains an antigen-binding moiety thatspecifically binds a tumor specific antigen or a tumor associatedantigen. In some embodiments, the engineered T-cell is a CAR (chimericantigen receptor) T-cell that contains an antigen-binding domain (e.g.scFv) that specifically binds to an antigen, such as a tumor specificantigen or tumor associated antigen. In some embodiments, theantigen-binding domain (e.g. scFv) is specific for CD19. Exemplary of aCAR is an anti-CD19 CAR, such as a CAR containing an anti-CD19 scFv setforth in SEQ ID NO:245. In some embodiments, the TIP protein isexpressed in an engineered T-cell receptor cell or and engineeredchimeric antigen receptor cell. In such embodiments, the engineered cellco-expresses the TIP and the CAR or TCR.

In some embodiments, the CAR further contains a spacer or hinge, atransmembrane domain, and an intracellular signaling domain (endodomain)comprising an ITAM signaling domain, such as a CD3zeta signaling domain.In some embodiments, the CAR further includes a costimulatory signalingdomain. In some embodiments, the spacer or hinge is present between theantigen-binding domain and the transmembrane domain, such as is betweenthe antigen-binding domain and plasma membrane when expressed on a cell.In some embodiments, the spacer or hinge is derived from IgG subclass(such as IgG1 and IgG4, IgD or CD8 (see e.g., Qin et al. (2017) J.Hematol. Oncol., 10:68). In some embodiments, the spacer or hinge isderived from IgG1.

In some embodiments, the spacer and transmembrane domain are the hingeand transmembrane domain derived from CD8, such as set forth in SEQ IDNO: 246 or 399 or a sequence of amino acids that exhibits at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity to SEQ ID NO:332 or 364. In some embodiments, theendodomain comprises at CD3-zeta signaling domain. In some embodiments,the CD3-zeta signaling domain comprises the sequence of amino acids setforth in SEQ ID NO: 247 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% to SEQ ID NO:247 and retains the activity of T cellsignaling. In some embodiments, the endodomain of a CAR-relatedtransmembrane immunomodulatory protein can further comprise acostimulatory signaling domain to further modulate immunomodulatoryresponses of the T-cell. In some embodiments, the costimulatorysignaling domain is CD28, ICOS, 41BB or OX40. In some embodiments, thecostimulatory signaling domain is a derived from CD28 or 4-1BB andcomprises the sequence of amino acids set forth in any of SEQ ID NOS:400-403 or a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQID NO:400-403 and retains the activity of T cell costimulatorysignaling.

In another embodiment, the engineered T-cell possesses a TCR, includinga recombinant or engineered TCR. In some embodiments, the TCR can be anative TCR. Those of skill in the art will recognize that generallynative mammalian T-cell receptors comprise an alpha and a beta chain (ora gamma and a delta chain) involved in antigen specific recognition andbinding. In some embodiments, the TCR is an engineered TCR that ismodified. In some embodiments, the TCR of an engineered T-cellspecifically binds to a tumor associated or tumor specific antigenpresented by an APC.

In some embodiments, the immunomodulatory polypeptides, such astransmembrane immunomodulatory polypeptides or secretableimmunomodulatory polypeptides, can be incorporated into engineeredcells, such as engineered T cells or engineered APCs, by a variety ofstrategies such as those employed for recombinant host cells. A varietyof methods to introduce a DNA construct into primary T cells are knownin the art. In some embodiments, viral transduction or plasmidelectroporation are employed. In typical embodiments, the nucleic acidmolecule encoding the immunomodulatory protein, or the expressionvector, comprises a signal peptide that localizes the expressedtransmembrane immunomodulatory proteins to the cellular membrane or forsecretion. In some embodiments, a nucleic acid encoding a transmembraneimmunomodulatory proteins of the invention is sub-cloned into a viralvector, such as a retroviral vector, which allows expression in the hostmammalian cell. The expression vector can be introduced into a mammalianhost cell and, under host cell culture conditions, the immunomodulatoryprotein is expressed on the surface or is secreted.

In an exemplary example, primary T-cells can be purified ex vivo (CD4cells or CD8 cells or both) and stimulated with an activation protocolconsisting of various TCR/CD28 agonists, such as anti-CD3/anti-CD28coated beads. After a 2 or 3 day activation process, a recombinantexpression vector containing an immunomodulatory polypeptide can bestably introduced into the primary T cells through art standardlentiviral or retroviral transduction protocols or plasmidelectroporation strategies. Cells can be monitored for immunomodulatorypolypeptide expression by, for example, flow cytometry usinganti-epitope tag or antibodies that cross-react with native parentalmolecule and polypeptides comprising variant CD80. T-cells that expressthe immunomodulatory polypeptide can be enriched through sorting withanti-epitope tag antibodies or enriched for high or low expressiondepending on the application.

Upon immunomodulatory polypeptide expression the engineered T-cell canbe assayed for appropriate function by a variety of means. Theengineered CAR or TCR co-expression can be validated to show that thispart of the engineered T cell was not significantly impacted by theexpression of the immunomodulatory protein. Once validated, standard invitro cytotoxicity, proliferation, or cytokine assays (e.g., IFN-gammaexpression) can be used to assess the function of engineered T-cells.Exemplary standard endpoints are percent lysis of the tumor line,proliferation of the engineered T-cell, or IFN-gamma protein expressionin culture supernatants. An engineered construct which results instatistically significant increased lysis of tumor line, increasedproliferation of the engineered T-cell, or increased IFN-gammaexpression over the control construct can be selected for. Additionally,non-engineered, such as native primary or endogenous T-cells could alsobe incorporated into the same in vitro assay to measure the ability ofthe immunomodulatory polypeptide construct expressed on the engineeredcells, such as engineered T-cells, to modulate activity, including, insome cases, to activate and generate effector function in bystander,native T-cells. Increased expression of activation markers such as CD69,CD44, or CD62L could be monitored on endogenous T cells, and increasedproliferation and/or cytokine production could indicate desired activityof the immunomodulatory protein expressed on the engineered T cells.

In some embodiments, the similar assays can be used to compare thefunction of engineered T cells containing the CAR or TCR alone to thosecontaining the CAR or TCR and a TIP construct. Typically, these in vitroassays are performed by plating various ratios of the engineered T celland a “tumor” cell line containing the cognate CAR or TCR antigentogether in culture. Standard endpoints are percent lysis of the tumorline, proliferation of the engineered T cell, or IFN-gamma production inculture supernatants. An engineered immunomodulatory protein whichresulted in statistically significant increased lysis of tumor line,increased proliferation of the engineered T cell, or increased IFN-gammaproduction over the same TCR or CAR construct alone can be selected for.Engineered human T cells can be analyzed in immunocompromised mice, likethe NSG strain, which lacks mouse T, NK and B cells. Engineered human Tcells in which the CAR or TCR binds a target counter-structure on thexenograft and is co-expressed with the TIP affinity modified IgSF domaincan be adoptively transferred in vivo at different cell numbers andratios compared to the xenograft. For example, engraftment of CD19+leukemia tumor lines containing a luciferase/GFP vector can be monitoredthrough bioluminescence or ex vivo by flow cytometry. In a commonembodiment, the xenograft is introduced into the murine model, followedby the engineered T cells several days later. Engineered T cellscontaining the immunomodulatory protein can be assayed for increasedsurvival, tumor clearance, or expanded engineered T cells numbersrelative to engineered T cells containing the CAR or TCR alone. As inthe in vitro assay, endogenous, native (i.e., non-engineered) human Tcells could be co-adoptively transferred to look for successful epitopespreading in that population, resulting in better survival or tumorclearance.

E. Infectious Agents Expressing Variant Polypeptides andImmunomodulatory Proteins

Also provided are infectious agents that contain nucleic acids encodingany of the variant polypeptides, such as CD80 vIgD polypeptides,including secretable or transmembrane immunomodulatory proteinsdescribed herein. In some embodiments, such infectious agents candeliver the nucleic acids encoding the variant immunomodulatorypolypeptides described herein, such as CD80 vIgD polypeptides, to atarget cell in a subject, e.g., immune cell and/or antigen-presentingcell (APC) or tumor cell in a subject. Also provided are nucleic acidscontained in such infectious agents, and/or nucleic acids for generationor modification of such infectious agents, such as vectors and/orplasmids, and compositions containing such infectious agents.

In some embodiments, the infectious agent is a microorganism or amicrobe. In some embodiments, the infectious agent is a virus or abacterium. In some embodiments, the infectious agent is a virus. In someembodiments, the infectious agent is a bacterium. In some embodiments,such infectious agents can deliver nucleic acid sequences encoding anyof the variant polypeptides, such as CD80 vIgD polypeptides, includingsecretable or transmembrane immunomodulatory proteins, described herein.Thus, in some embodiments, the cell in a subject that is infected orcontacted by the infectious agents can be rendered to express on thecell surface or secrete, the variant immunomodulatory polypeptides. Insome embodiments, the infectious agent can also deliver one or moreother therapeutics or nucleic acids encoding other therapeutics to thecell and/or to an environment within the subject. In some embodiments,other therapeutics that can be delivered by the infectious agentsinclude cytokines or other immunomodulatory molecules.

In some embodiments, the infectious agent, e.g., virus or bacteria,contains nucleic acid sequences that encode any of the variantpolypeptides, such as CD80 vIgD polypeptides, including secretable ortransmembrane immunomodulatory proteins, described herein, and by virtueof contact and/or infection of a cell in the subject, the cell expressesthe variant polypeptides, such as CD80 vIgD polypeptides, includingsecretable or transmembrane immunomodulatory proteins, encoded by thenucleic acid sequences contained in the infectious agent. In someembodiments, the infectious agent can be administered to the subject. Insome embodiments, cells from the subject can be transduced by theinfectious agent ex vivo.

In some embodiments, the variant polypeptides, such as CD80 vIgDpolypeptides, including transmembrane immunomodulatory proteins,expressed by the cell infected by the infectious agent is atransmembrane protein and is surface expressed. In some embodiments, thevariant polypeptides, such as CD80 vIgD polypeptides, includingsecretable immunomodulatory proteins, expressed by the cell infected bythe infectious agent is expressed and secreted from the cell. Thetransmembrane immunomodulatory protein or secreted immunomodulatoryprotein can be any described herein.

In some embodiments, the cells in the subject that are targeted by theinfectious agent include a tumor cell, an immune cell, and/or anantigen-presenting cell (APC). In some embodiments, the infectious agenttargets a cell in the tumor microenvironment (TME). In some embodiments,the infectious agent delivers the nucleic acids encoding the variantpolypeptides, such as CD80 vIgD polypeptides, including secretable ortransmembrane immunomodulatory proteins, to an appropriate cell (forexample, an APC, such as a cell that displays a peptide/MHC complex onits cell surface, such as a dendritic cell) or tissue (e.g., lymphoidtissue) that will induce and/or augment the desired effect, e.g.,immunomodulation and/or a specific cell-medicated immune response, e.g.,CD4 and/or CD8 T cell response, which CD8 T cell response may include acytotoxic T cell (CTL) response. In some embodiments, the infectiousagent targets an APC, such as a dendritic cell (DC). In someembodiments, the nucleic acid molecule delivered by the infectiousagents described herein include appropriate nucleic acid sequencesnecessary for the expression of the operably linked coding sequencesencoding the variant immunomodulatory polypeptides, in a particulartarget cell, e.g., regulatory elements such as promoters.

In some embodiments, the infectious agent that contains nucleic acidsequences encoding the immunomodulatory polypeptides can also containnucleic acid sequences that encode one or more additional gene products,e.g., cytokines, prodrug converting enzymes, cytotoxins and/ordetectable gene products. For example, in some embodiments, theinfectious agent is an oncolytic virus and the virus can include nucleicacid sequences encoding additional therapeutic gene products (see, e.g.,Kirn et al., (2009) Nat Rev Cancer 9:64-71; Garcia-Aragoncillo et al.,(2010) Curr Opin Mol Ther 12:403-411; see U.S. Pat. Nos. 7,588,767,7,588,771, 7,662,398 and 7,754,221 and U.S. Pat. Publ. Nos.2007/0202572, 2007/0212727, 2010/0062016, 2009/0098529, 2009/0053244,2009/0155287, 2009/0117034, 2010/0233078, 2009/0162288, 2010/0196325,2009/0136917 and 2011/0064650. In some embodiments, the additional geneproduct can be a therapeutic gene product that can result in death ofthe target cell (e.g., tumor cell) or gene products that can augment orboost or regulate an immune response (e.g., cytokine). Exemplary geneproducts also include among an anticancer agent, an anti-metastaticagent, an antiangiogenic agent, an immunomodulatory molecule, an immunecheckpoint inhibitor, an antibody, a cytokine, a growth factor, anantigen, a cytotoxic gene product, a pro-apoptotic gene product, ananti-apoptotic gene product, a cell matrix degradative gene, genes fortissue regeneration and reprogramming human somatic cells topluripotency, and other genes described herein or known to one of skillin the art. In some embodiments, the additional gene product isGranulocyte-macrophage colony-stimulating factor (GM-CSF).

1. Viruses

In some embodiments, the infectious agent is a virus. In someembodiments, the infectious agent is an oncolytic virus, or a virus thattargets particular cells, e.g., immune cells. In some embodiments, theinfectious agent targets a tumor cell and/or cancer cell in the subject.In some embodiments, the infectious agent targets an immune cell or anantigen-presenting cell (APC).

In some embodiments, the infectious agent is an oncolytic virus.Oncolytic viruses are viruses that accumulate in tumor cells andreplicate in tumor cells. By virtue of replication in the cells, andoptional delivery of nucleic acids encoding variant immunomodulatoryvariant CD80 polypeptides or immunomodulatory proteins described herein,tumor cells are lysed, and the tumor shrinks and can be eliminated.Oncolytic viruses can also have a broad host and cell type range. Forexample, oncolytic viruses can accumulate in immunoprivileged cells orimmunoprivileged tissues, including tumors and/or metastases, and alsoincluding wounded tissues and cells, thus allowing the delivery andexpression of nucleic acids encoding the variant immunomodulatorypolypeptides described herein in a broad range of cell types. Oncolyticviruses can also replicate in a tumor cell specific manner, resulting intumor cell lysis and efficient tumor regression.

Exemplary oncolytic viruses include adenoviruses, adeno-associatedviruses, herpes viruses, Herpes Simplex Virus, Vesticular Stomaticvirus, Reovirus, Newcastle Disease virus, parvovirus, measles virus,vesticular stomatitis virus (VSV), Coxsackie virus and Vaccinia virus.In some embodiments, oncolytic viruses can specifically colonize solidtumors, while not infecting other organs, and can be used as aninfectious agent to deliver the nucleic acids encoding the variantimmunomodulatory polypeptides described herein to such solid tumors.

Oncolytic viruses for use in delivering the nucleic acids encodingvariant CD80 polypeptides or immunomodulatory proteins described herein,can be any of those known to one of skill in the art and include, forexample, vesicular stomatitis virus, see, e.g., U.S. Pat. Nos.7,731,974, 7,153,510, 6,653,103 and U.S. Pat. Pub. Nos. 2010/0178684,2010/0172877, 2010/0113567, 2007/0098743, 20050260601, 20050220818 andEP Pat. Nos. 1385466, 1606411 and 1520175; herpes simplex virus, see,e.g., U.S. Pat. Nos. 7,897,146, 7,731,952, 7,550,296, 7,537,924,6,723,316, 6,428,968 and U.S. Pat. Pub. Nos. 2014/0154216, 2011/0177032,2011/0158948, 2010/0092515, 2009/0274728, 2009/0285860, 2009/0215147,2009/0010889, 2007/0110720, 2006/0039894, 2004/0009604, 2004/0063094,International Patent Pub. Nos., WO 2007/052029, WO 1999/038955;retroviruses, see, e.g., U.S. Pat. Nos. 6,689,871, 6,635,472, 5,851,529,5,716,826, 5,716,613 and U.S. Pat. Pub. No. 20110212530; vacciniaviruses, see, e.g., 2016/0339066, and adeno-associated viruses, see,e.g., U.S. Pat. Nos. 8,007,780, 7,968,340, 7,943,374, 7,906,111,7,927,585, 7,811,814, 7,662,627, 7,241,447, 7,238,526, 7,172,893,7,033,826, 7,001,765, 6,897,045, and 6,632,670.

Oncolytic viruses also include viruses that have been geneticallyaltered to attenuate their virulence, to improve their safety profile,enhance their tumor specificity, and they have also been equipped withadditional genes, for example cytotoxins, cytokines, prodrug convertingenzymes to improve the overall efficacy of the viruses (see, e.g., Kimet al., (2009) Nat Rev Cancer 9:64-71; Garcia-Aragoncillo et al., (2010)Curr Opin Mol Ther 12:403-411; see U.S. Pat. Nos. 7,588,767, 7,588,771,7,662,398 and 7,754,221 and U.S. Pat. Publ. Nos. 2007/0202572,2007/0212727, 2010/0062016, 2009/0098529, 2009/0053244, 2009/0155287,2009/0117034, 2010/0233078, 2009/0162288, 2010/0196325, 2009/0136917 and2011/0064650). In some embodiments, the oncolytic viruses can be thosethat have been modified so that they selectively replicate in cancerouscells, and, thus, are oncolytic. For example, the oncolytic virus is anadenovirus that has been engineered to have modified tropism for tumortherapy and also as gene therapy vectors. Exemplary of such is ONYX-015,H101 and Ad5ΔCR (Hallden and Portella (2012) Expert Opin Ther Targets,16:945-58) and TNFerade (McLoughlin et al. (2005) Ann. Surg. Oncol.,12:825-30), or a conditionally replicative adenovirus Oncorine®.

In some embodiments, the infectious agent is a modified herpes simplexvirus. In some embodiments, the infectious agent is a modified versionof Talimogene laherparepvec (also known as T-Vec, Imlygic or OncoVexGM-CSF), that is modified to contain nucleic acids encoding any of thevariant immunomodulatory polypeptides described herein, such as any ofthe variant CD80 polypeptides or immunomodulatory proteins describedherein. In some embodiments, the infectious agent is a modified herpessimplex virus that is described, e.g., in WO 2007/052029, WO1999/038955, US 2004/0063094, US 2014/0154216, or, variants thereof.

In some embodiments, the infectious agent is a virus that targets aparticular type of cells in a subject that is administered the virus,e.g., a virus that targets immune cells or antigen-presenting cells(APCs). Dendritic cells (DCs) are essential APCs for the initiation andcontrol of immune responses. DCs can capture and process antigens,migrate from the periphery to a lymphoid organ, and present the antigensto resting T cells in a major histocompatibility complex(MHC)-restricted fashion. In some embodiments, the infectious agent is avirus that specifically can target DCs to deliver nucleic acids encodingthe variant CD80 polypeptides or immunomodulatory proteins forexpression in DCs. In some embodiments, the virus is a lentivirus or avariant thereof. In some embodiments, the virus is a lentivirus that ispseudotyped to efficiently bind to and productively infect cellsexpressing the cell surface marker dendritic cell-specific intercellularadhesion molecule-3-grabbing non-integrin (DC-SIGN), such as DCs. Insome embodiments, the virus is a lentivirus pseudotyped with a Sindbisvirus E2 glycoprotein, such as those described in WO 2013/149167. Insome embodiments, the virus allows for delivery and expression of asequence of interest (e.g., a nucleic acid encoding any of the variantCD80 polypeptides or immunomodulatory proteins described herein) to aDC. In some embodiments, the virus includes those described in WO2008/011636 or US 2011/0064763, or variants thereof

2. Bacteria

In some embodiments, the infectious agent is a bacterium. For example,in some embodiments, the bacteria can deliver nucleic acids encoding anyof the variant immunomodulatory polypeptides described herein, e.g.,variant CD80 polypeptide or immunomodulatory protein, to a target cellin the subject, such as a tumor cell, an immune cell, anantigen-presenting cell and/or a phagocytic cell. In some embodiments,the bacterium can be preferentially targeted to a specific environmentwithin a subject, such as a tumor microenvironment (TME), for expressionand/or secretion of the variant immunomodulatory polypeptides and/or totarget specific cells in the environment for expression of the variantimmunomodulatory polypeptides.

In some embodiments, the bacterium delivers the nucleic acids to thecells via bacterial-mediated transfer of plasmid DNA to mammalian cells(also referred to as “bactofection”). For example, in some embodiments,delivery of genetic material is achieved through entry of the entirebacterium into target cells. In some embodiments, spontaneous or inducedbacterial lysis can lead to the release of plasmid for subsequenteukaryotic cell expression. In some embodiments, the bacterium candeliver nucleic acids to non-phagocytic mammalian cells (e.g., tumorcells) and/or to phagocytic cells, e.g., certain immune cells and/orAPCs. In some embodiments, the nucleic acids delivered by the bacteriumcan be transferred to the nucleus of the cell in the subject forexpression. In some embodiments, the nucleic acids also includeappropriate nucleic acid sequences necessary for the expression of theoperably linked sequences encoding the variant immunomodulatorypolypeptides in a particular host cell, e.g., regulatory elements suchas promoters or enhancers. In some embodiments, the infectious agentthat is a bacterium can deliver nucleic acids encoding theimmunomodulatory proteins in the form of an RNA, such as a pre-madetranslation-competent RNA delivered to the cytoplasm of the target cellfor translation by the target cell's machinery.

In some embodiments, the bacterium can replicate and lyse the targetcells, e.g., tumor cells. In some embodiments, the bacterium can containand/or release nucleic acid sequences and/or gene products in thecytoplasm of the target cells, thereby killing the target cell, e.g.,tumor cell. In some embodiments, the infectious agent is bacterium thatcan replicate specifically in a particular environment in the subject,e.g., tumor microenvironment (TME). For example, in some embodiments,the bacterium can replicate specifically in anaerobic or hypoxicmicroenvironments. In some embodiments, conditions or factors present inparticular environments, e.g., aspartate, serine, citrate, ribose orgalactose produced by cells in the TME, can act as chemoattractants toattract the bacterium to the environment. In some embodiments, thebacterium can express and/or secrete the immunomodulatory proteinsdescribed herein in the environment, e.g., TME.

In some embodiments, the infectious agent is a bacterium that is aListeria sp., a Bifidobacterium sp., an Escherichia sp., a Closteridiumsp., a Salmonella sp., a Shigella sp., a Vibrio sp. or a Yersinia sp. Insome embodiments, the bacterium is selected from among one or more ofListeria monocytogenes, Salmonella typhimurium, Salmonella choleraesuis,Escherichia coli, Vibrio cholera, Clostridium perfringens, Clostridiumbutyricum, Clostridium novyi, Clostridium acetobutylicum,Bifidobacterium infantis, Bifidobacterium longum and Bifidobacteriumadolescentis. In some embodiments, the bacterium is an engineeredbacterium. In some embodiments, the bacterium is an engineered bacteriumsuch as those described in, e.g., Seow and Wood (2009) Molecular Therapy17(5):767-777; Baban et al. (2010) Bioengineered Bugs 1:6, 385-394;Patyar et al. (2010) J Biomed Sci 17:21; Tangney et al. (2010)Bioengineered Bugs 1:4, 284-287; van Pijkeren et al. (2010) Hum GeneTher. 21(4):405-416; WO 2012/149364; WO 2014/198002; U.S. Pat. Nos.9,103,831; 9,453,227; US 2014/0186401; US 2004/0146488; US 2011/0293705;US 2015/0359909 and EP 3020816. The bacterium can be modified to delivernucleic acid sequences encoding any of the variant immunomodulatorypolypeptides, conjugates and/or fusions provided herein, and/or toexpress such variant immunomodulatory polypeptides in the subject.

F. Nucleic Acids, Vectors and Methods for Producing the Polypeptides orCells

Provided herein are isolated or recombinant nucleic acids collectivelyreferred to as “nucleic acids” which encode any of the various providedembodiments of the variant CD80 polypeptides or immunomodulatorypolypeptides provided herein. In some embodiments, nucleic acidsprovided herein, including all described below, are useful inrecombinant production (e.g., expression) of variant CD80 polypeptidesor immunomodulatory polypeptides provided herein. In some embodiments,nucleic acids provided herein, including all described below, are usefulin expression of variant CD80 polypeptides or immunomodulatorypolypeptides provided herein in cells, such as in engineered cells, e.g.immune cells, or infectious agent cells. The nucleic acids providedherein can be in the form of RNA or in the form of DNA, and includemRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. The nucleicacids provided herein are typically DNA molecules, and usuallydouble-stranded DNA molecules. However, single-stranded DNA,single-stranded RNA, double-stranded RNA, and hybrid DNA/RNA nucleicacids or combinations thereof comprising any of the nucleotide sequencesof the invention also are provided.

Also provided herein are recombinant expression vectors and recombinanthost cells useful in producing the variant CD80 polypeptides orimmunomodulatory polypeptides provided herein.

Also provided herein are engineered cells, such as engineered immunecells, containing any of the provided nucleic acid molecules or theencoded variant CD80 polypeptides or immunomodulatory polypeptides, suchas any of the transmembrane immunomodulatory polypeptides or secretableimmunomodulatory polypeptides.

Also provided herein are infectious agents, such as bacterial or viralcells, containing any of the provided nucleic acid molecules or theencoded variant CD80 polypeptides or immunomodulatory polypeptides, suchas any of the transmembrane immunomodulatory polypeptides or secretableimmunomodulatory polypeptides.

In any of the above provided embodiments, the nucleic acids encoding theimmunomodulatory polypeptides provided herein can be introduced intocells using recombinant DNA and cloning techniques. To do so, arecombinant DNA molecule encoding a immunomodulatory polypeptide isprepared. Methods of preparing such DNA molecules are well known in theart. For instance, sequences coding for the peptides could be excisedfrom DNA using suitable restriction enzymes. Alternatively, the DNAmolecule could be synthesized using chemical synthesis techniques, suchas the phosphoramidite method. Also, a combination of these techniquescould be used. In some instances, a recombinant or synthetic nucleicacid may be generated through polymerase chain reaction (PCR). In someembodiments, a DNA insert can be generated encoding one or more variantCD80 polypeptides containing at least one affinity-modified IgSF domainand, in some embodiments, a signal peptide, a transmembrane domainand/or an endodomain in accord with the provided description. This DNAinsert can be cloned into an appropriate transduction/transfectionvector as is known to those of skill in the art. Also provided areexpression vectors containing the nucleic acid molecules.

In some embodiments, the expression vectors are capable of expressingthe immunomodulatory proteins in an appropriate cell under conditionssuited to expression of the protein. In some aspects, nucleic acidmolecule or an expression vector comprises the DNA molecule that encodesthe immunomodulatory protein operatively linked to appropriateexpression control sequences. Methods of effecting this operativelinking, either before or after the DNA molecule is inserted into thevector, are well known. Expression control sequences include promoters,activators, enhancers, operators, ribosomal binding sites, startsignals, stop signals, cap signals, polyadenylation signals, and othersignals involved with the control of transcription or translation.

In some embodiments, expression of the immunomodulatory protein iscontrolled by a promoter or enhancer to control or regulate expression.The promoter is operably linked to the portion of the nucleic acidmolecule encoding the variant polypeptide or immunomodulatory protein.In some embodiments, the promotor is a constitutively active promotor(such as a tissue-specific constitutively active promotor or otherconstitutive promotor). In some embodiments, the promotor is aninducible promotor, which may be responsive to an inducing agent (suchas a T cell activation signal).

In some embodiments, a constitutive promoter is operatively linked tothe nucleic acid molecule encoding the variant polypeptide orimmunomodulatory protein. Exemplary constitutive promoters include theSimian vacuolating virus 40 (SV40) promoter, the cytomegalovirus (CMV)promoter, the ubiquitin C (UbC) promoter, and the EF-1 alpha (EF1a)promoter. In some embodiments, the constitutive promoter is tissuespecific. For example, in some embodiments, the promoter allows forconstitutive expression of the immunomodulatory protein in specifictissues, such as immune cells, lymphocytes, or T cells. Exemplarytissue-specific promoters are described in U.S. Pat. No. 5,998,205,including, for example, a fetoprotein, DF3, tyrosinase, CEA, surfactantprotein, and ErbB2 promoters.

In some embodiments, an inducible promoter is operatively linked to thenucleic acid molecule encoding the variant polypeptide orimmunomodulatory protein such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. For example, the promoter can be a regulatedpromoter and transcription factor expression system, such as thepublished tetracycline-regulated systems or other regulatable systems(see, e.g. published International PCT Appl. No. WO 01/30843), to allowregulated expression of the encoded polypeptide. An exemplaryregulatable promoter system is the Tet-On (and Tet-Off) systemavailable, for example, from Clontech (Palo Alto, Calif.). This promotersystem allows the regulated expression of the transgene controlled bytetracycline or tetracycline derivatives, such as doxycycline. Otherregulatable promoter systems are known (see e.g., published U.S.Application No. 2002-0168714, entitled “Regulation of Gene ExpressionUsing Single-Chain, Monomeric, Ligand Dependent Polypeptide Switches,”which describes gene switches that contain ligand binding domains andtranscriptional regulating domains, such as those from hormonereceptors).

In some embodiments, the promotor is responsive to an element responsiveto T-cell activation signaling. Solely by way of example, in someembodiments, an engineered T cell comprises an expression vectorencoding the immunomodulatory protein and a promotor operatively linkedto control expression of the immunomodulatory protein. The engineered Tcell can be activated, for example by signaling through an engineered Tcell receptor (TCR) or a chimeric antigen rector (CAR), and therebytriggering expression and secretion of the immunomodulatory proteinthrough the responsive promotor.

In some embodiments, an inducible promoter is operatively linked to thenucleic acid molecule encoding the immunomodulatory protein such thatthe immunomodulatory protein is expressed in response to a nuclearfactor of activated T-cells (NFAT) or nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB). For example, in some embodiments,the inducible promoter comprises a binding site for NFAT or NF-κB. Forexample, in some embodiments, the promoter is an NFAT or NF-κB promoteror a functional variant thereof. Thus, in some embodiments, the nucleicacids make it possible to control the expression of immunomodulatoryprotein while also reducing or eliminating the toxicity of theimmunomodulatory protein. In particular, engineered immune cellscomprising the nucleic acids of the invention express and secrete theimmunomodulatory protein only when the cell (e.g., a T-cell receptor(TCR) or a chimeric antigen receptor (CAR) expressed by the cell) isspecifically stimulated by an antigen and/or the cell (e.g., the calciumsignaling pathway of the cell) is non-specifically stimulated by, e.g.,phorbol myristate acetate (PMA)/Ionomycin. Accordingly, the expressionand, in some cases, secretion, of immunomodulatory protein can becontrolled to occur only when and where it is needed (e.g., in thepresence of an infectious disease-causing agent, cancer, or at a tumorsite), which can decrease or avoid undesired immunomodulatory proteininteractions.

In some embodiments, the nucleic acid encoding an immunomodulatoryprotein described herein comprises a suitable nucleotide sequence thatencodes a NFAT promoter, NF-κB promoter, or a functional variant thereof“NFAT promoter” as used herein means one or more NFAT responsiveelements linked to a minimal promoter. “NF-κB promoter” refers to one ormore NF-κB responsive elements linked to a minimal promoter. In someembodiments, the minimal promoter of a gene is a minimal human IL-2promoter or a CMV promoter. The NFAT responsive elements may comprise,e.g., NFAT1, NFAT2, NFAT3, and/or NFAT4 responsive elements. The NFATpromoter, NF-κB promoter, or a functional variant thereof may compriseany number of binding motifs, e.g., at least two, at least three, atleast four, at least five, or at least six, at least seven, at leasteight, at least nine, at least ten, at least eleven, or up to twelvebinding motifs.

The resulting recombinant expression vector having the DNA moleculethereon is used to transform an appropriate host. This transformationcan be performed using methods well known in the art. In someembodiments, a nucleic acid provided herein further comprises nucleotidesequence that encodes a secretory or signal peptide operably linked tothe nucleic acid encoding an immunomodulatory polypeptide such that aresultant soluble immunomodulatory polypeptide is recovered from theculture medium, host cell, or host cell periplasm. In other embodiments,the appropriate expression control signals are chosen to allow formembrane expression of an immunomodulatory polypeptide. Furthermore,commercially available kits as well as contract manufacturing companiescan also be utilized to make engineered cells or recombinant host cellsprovided herein.

In some embodiments, the resulting expression vector having the DNAmolecule thereon is used to transform, such as transduce, an appropriatecell. The introduction can be performed using methods well known in theart. Exemplary methods include those for transfer of nucleic acidsencoding the receptors, including via viral, e.g., retroviral orlentiviral, transduction, transposons, and electroporation. In someembodiments, the expression vector is a viral vector. In someembodiments, the nucleic acid is transferred into cells by lentiviral orretroviral transduction methods.

Any of a large number of publicly available and well-known mammalianhost cells, including mammalian T-cells or APCs, can be used in thepreparing the polypeptides or engineered cells. The selection of a cellis dependent upon a number of factors recognized by the art. Theseinclude, for example, compatibility with the chosen expression vector,toxicity of the peptides encoded by the DNA molecule, rate oftransformation, ease of recovery of the peptides, expressioncharacteristics, bio-safety and costs. A balance of these factors mustbe struck with the understanding that not all cells can be equallyeffective for the expression of a particular DNA sequence.

In some embodiments, the host cells can be a variety of eukaryoticcells, such as in yeast cells, or with mammalian cells such as Chinesehamster ovary (CHO) or HEK293 cells. Host cells can also be prokaryoticcells, such as with E. coli. The transformed recombinant host iscultured under polypeptide expressing conditions, and then purified toobtain a soluble protein. Recombinant host cells can be cultured underconventional fermentation conditions so that the desired polypeptidesare expressed. Such fermentation conditions are well known in the art.Finally, the polypeptides provided herein can be recovered and purifiedfrom recombinant cell cultures by any of a number of methods well knownin the art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, and affinitychromatography. Protein refolding steps can be used, as desired, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed in the finalpurification steps.

In some embodiments, the cell is an immune cells, such as any describedabove in connection with preparing engineered cells. In someembodiments, such engineered cells are primary cells. In someembodiments, the engineered cells are autologous to the subject. In someembodiment, the engineered cells are allogeneic to the subject. In someembodiments, the engineered cells are obtained from a subject, such asby leukapheresis, and transformed ex vivo for expression of theimmunomodulatory polypeptide, e.g. transmembrane immunomodulatorypolypeptide or secretable immunomodulatory polypeptide.

Also provided are nucleic acids encoding any of the variantimmunomodulatory polypeptides contained in infectious agents describedherein. In some embodiments, the infectious agents deliver the nucleicacids to a cell in the subject, and/or permit expression of the encodedvariant polypeptides in the cell. Also provided are nucleic acids thatare used to generate, produce or modify such infectious agents. Forexample, in some embodiments, provided are vectors and/or plasmids thatcontain nucleic acids encoding the variant immunomodulatorypolypeptides, for generation of the infectious agents, delivery to thecells in a subject and/or expression of the variant immunomodulatorypolypeptides in the cells in the subject.

In some embodiments, the provided nucleic acids are recombinant viral orbacterial vectors containing nucleic acid sequences encoding the variantimmunomodulatory polypeptides. In some embodiments, the recombinantvectors can be used to produce an infectious agent that contains nucleicacid sequences encoding the variant immunomodulatory polypeptides and/orto be delivered to a target cell in the subject for expression by thetarget cell. In some embodiments, the recombinant vector is anexpression vector. In some embodiments, the recombinant vector includesappropriate sequences necessary for generation and/or production of theinfectious agent and expression in the target cell.

In some embodiments, the recombinant vector is a plasmid or cosmid.Plasmid or cosmid containing nucleic acid sequences encoding the variantimmunomodulatory polypeptides, as described herein, is readilyconstructed using standard techniques well known in the art. Forgeneration of the infectious agent, the vector or genome can beconstructed in a plasmid form that can then be transfected into apackaging or producer cell line or a host bacterium. The recombinantvectors can be generated using any of the recombinant techniques knownin the art. In some embodiments, the vectors can include a prokaryoticorigin of replication and/or a gene whose expression confers adetectable or selectable marker such as a drug resistance forpropagation and/or selection in prokaryotic systems.

In some embodiments, the recombinant vector is a viral vector. Exemplaryrecombinant viral vectors include a lentiviral vector genome, poxvirusvector genome, vaccinia virus vector genome, adenovirus vector genome,adenovirus-associated virus vector genome, herpes virus vector genome,and alpha virus vector genome. Viral vectors can be live, attenuated,replication conditional or replication deficient, non-pathogenic(defective), replication competent viral vector, and/or is modified toexpress a heterologous gene product, e.g., the variant immunomodulatorypolypeptides provided herein. Vectors for generation of viruses also canbe modified to alter attenuation of the virus, which includes any methodof increasing or decreasing the transcriptional or translational load.

Exemplary viral vectors that can be used include modified vaccinia virusvectors (see, e.g., Guerra et al., J. Virol. 80:985-98 (2006); Tartagliaet al., AIDS Research and Human Retroviruses 8: 1445-47 (1992); Gheradiet al., J. Gen. Virol. 86:2925-36 (2005); Mayr et al., Infection 3:6-14(1975); Hu et al., J. Virol. 75: 10300-308 (2001); U.S. Pat. Nos.5,698,530, 6,998,252, 5,443,964, 7,247,615 and 7,368,116); adenovirusvector or adenovirus-associated virus vectors (see, e.g., Molin et al.,J. Virol. 72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol.Biol. 19:936-41 (1998); Mercier et al., Proc. Natl. Acad. Sci. USA101:6188-93 (2004); U.S. Pat. Nos. 6,143,290; 6,596,535; 6,855,317;6,936,257; 7,125,717; 7,378,087; 7,550,296); retroviral vectorsincluding those based upon murine leukemia virus (MuLV), gibbon apeleukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiencyvirus (SIV), human immunodeficiency virus (HIV), and combinations (see,e.g., Buchscher et al., J. Virol. 66:2731-39 (1992); Johann et al., J.Virol. 66: 1635-40 (1992); Sommerfelt et al., Virology 176:58-59 (1990);Wilson et al., J. Virol. 63:2374-78 (1989); Miller et al., J. Virol.65:2220-24 (1991); Miller et al., Mol. Cell Biol. 10:4239 (1990);Kolberg, NIH Res. 4:43 1992; Cornetta et al., Hum. Gene Ther. 2:215(1991)); lentiviral vectors including those based upon HumanImmunodeficiency Virus (HIV-1), HIV-2, feline immunodeficiency virus(FIV), equine infectious anemia virus, Simian Immunodeficiency Virus(SIV), and maedi/visna virus (see, e.g., Pfeifer et al., Annu. Rev.Genomics Hum. Genet. 2: 177-211 (2001); Zufferey et al., J. Virol. 72:9873, 1998; Miyoshi et al., J. Virol. 72:8150, 1998; Philpott andThrasher, Human Gene Therapy 18:8, 2007; Engelman et al., J. Virol. 69:2729, 1995; Nightingale et al., Mol. Therapy, 13: 1121, 2006; Brown etal., J. Virol. 73:9011 (1999); WO 2009/076524; WO 2012/141984; WO2016/011083; McWilliams et al., J. Virol. 77: 11150, 2003; Powell etal., J. Virol. 70:5288, 1996) or any, variants thereof, and/or vectorsthat can be used to generate any of the viruses described above. In someembodiments, the recombinant vector can include regulatory sequences,such as promoter or enhancer sequences, that can regulate the expressionof the viral genome, such as in the case for RNA viruses, in thepackaging cell line (see, e.g., U.S. Pat. Nos. 5,385,839 and 5,168,062).

In some embodiments, the recombinant vector is an expression vector,e.g., an expression vector that permits expression of the encoded geneproduct when delivered into the target cell, e.g., a cell in thesubject, e.g., a tumor cell, an immune cell and/or an APC. In someembodiments, the recombinant expression vectors contained in theinfectious agent are capable of expressing the immunomodulatory proteinsin the target cell in the subject, under conditions suited to expressionof the protein.

In some aspects, nucleic acids or an expression vector comprises anucleic acid sequence that encodes the immunomodulatory proteinoperatively linked to appropriate expression control sequences. Methodsof affecting this operative linking, either before or after the nucleicacid sequence encoding the immunomodulatory protein is inserted into thevector, are well known. Expression control sequences include promoters,activators, enhancers, operators, ribosomal binding sites, startsignals, stop signals, cap signals, polyadenylation signals, and othersignals involved with the control of transcription or translation. Thepromoter can be operably linked to the portion of the nucleic acidsequence encoding the immunomodulatory protein. In some embodiments, thepromotor is a constitutively active promotor in the target cell (such asa tissue-specific constitutively active promotor or other constitutivepromotor). For example, the recombinant expression vector may alsoinclude, lymphoid tissue-specific transcriptional regulatory elements(TRE) such as a B lymphocyte, T lymphocyte, or dendritic cell specificTRE. Lymphoid tissue specific TRE are known in the art (see, e.g.,Thompson et al., Mol. Cell. Biol. 12:1043-53 (1992); Todd et al., J.Exp. Med. 177:1663-74 (1993); Penix et al., J. Exp. Med. 178:1483-96(1993)). In some embodiments, the promotor is an inducible promotor,which may be responsive to an inducing agent (such as a T cellactivation signal). In some embodiments, nucleic acids delivered to thetarget cell in the subject, e.g., tumor cell, immune cell and/or APC,can be operably linked to any of the regulatory elements describedabove.

In some embodiments, the vector is a bacterial vector, e.g., a bacterialplasmid or cosmid. In some embodiments, the bacterial vector isdelivered to the target cell, e.g., tumor cells, immune cells and/orAPCs, via bacterial-mediated transfer of plasmid DNA to mammalian cells(also referred to as “bactofection”). In some embodiments, the deliveredbacterial vector also contains appropriate expression control sequencesfor expression in the target cells, such as a promoter sequence and/orenhancer sequences, or any regulatory or control sequences describedabove. In some embodiments, the bacterial vector contains appropriateexpression control sequences for expression and/or secretion of theencoded variant polypeptides in the infectious agent, e.g., thebacterium.

In some embodiments, polypeptides provided herein can also be made bysynthetic methods. Solid phase synthesis is the preferred technique ofmaking individual peptides since it is the most cost-effective method ofmaking small peptides. For example, well known solid phase synthesistechniques include the use of protecting groups, linkers, and solidphase supports, as well as specific protection and deprotection reactionconditions, linker cleavage conditions, use of scavengers, and otheraspects of solid phase peptide synthesis. Peptides can then be assembledinto the polypeptides as provided herein.

IV. METHODS OF ASSESSING ACTIVITY IMMUNE MODULATION OF VARIANT CD80POLYPEPTIDES AND IMMUNOMODULATORY PROTEINS

In some embodiments, the variant CD80 polypeptides provided herein (e.g.full-length and/or specific binding fragments or conjugates, stackconstructs or fusion thereof) exhibit immunomodulatory activity tomodulate T cell activation. In some embodiments, CD80 polypeptidesmodulate IFN-gamma expression in a primary T cell assay relative to awild-type or unmodified CD80 control. In some cases, modulation ofIFN-gamma expression can increase or decrease IFN-gamma expressionrelative to the control. Assays to determine specific binding andIFN-gamma expression are well-known in the art and include the MLR(mixed lymphocyte reaction) assays measuring interferon-gamma cytokinelevels in culture supernatants (Wang et al., Cancer Immunol Res. 2014Sep.: 2(9):846-56), SEB (staphylococcal enterotoxin B) T cellstimulation assay (Wang et al., Cancer Immunol Res. 2014 Sep.:2(9):846-56), and anti-CD3 T cell stimulation assays (Li and Kurlander,J Transl Med. 2010: 8: 104).

In some embodiments, a variant CD80 polypeptide can in some embodimentsincrease or, in alternative embodiments, decrease IFN-gamma(interferon-gamma) expression in a primary T-cell assay relative to awild-type CD80 control. In some embodiments of the provided polypeptidescontaining a soluble variant CD80 sequence, the polypeptide can increaseIFN-gamma expression and, in alternative embodiments, decrease IFN-gammaexpression in a primary T-cell assay relative to a wild-type CD80control. In some embodiments of the provided polypeptides containingmultiple variant CD80 sequences, the polypeptide can increase IFN-gammaexpression and, in alternative embodiments, decrease IFN-gammaexpression in a primary T-cell assay relative to a wild-type CD80control.

Those of skill will recognize that the format of the primary T-cellassay used to determine an increase in IFN-gamma expression can differfrom that employed to assay for a decrease in IFN-gamma expression. Inassaying for the ability of a variant CD80 to decrease IFN-gammaexpression in a primary T-cell assay, a Mixed Lymphocyte Reaction (MLR)assay can be used as described in Example 6. In some cases, a solubleform of a variant CD80 can be employed to determine the ability of thevariant CD80 to antagonize and thereby decrease the IFN-gamma expressionin a MLR as likewise described in Example 6.

Alternatively, in assaying for the ability of a variant CD80 to increaseIFN-gamma expression in a primary T-cell assay, a co-immobilizationassay can be used as described in Example 6. In a co-immobilizationassay, a TCR signal, provided in some embodiments by anti-CD3 antibody,is used in conjunction with a co-immobilized variant CD80 to determinethe ability to increase IFN-gamma expression relative to a CD80 control.In some cases, a soluble form of a variant CD80 that is multimerized toa degree to provide multivalent binding can be employed to determine theability of the variant CD80 to agonize and thereby increase theIFN-gamma expression in a MLR as likewise described in Example 6.

Use of proper controls is known to those of skill in the art, however,in the aforementioned embodiments, the control typically involves use ofthe unmodified CD80, such as a wild-type of native CD80 isoform from thesame mammalian species from which the variant CD80 was derived ordeveloped. Irrespective of whether the binding affinity to either one orboth of ICOS and CD28 is increased or decreased, a variant CD80 in someembodiments will increase IFN-gamma expression and, in alternativeembodiments, decrease IFN-gamma expression in a primary T-cell assayrelative to a wild-type CD80 control.

In some embodiments, a variant CD80 increases IFN-gamma expression(i.e., protein expression) relative to a wild-type or unmodified CD80control by at least: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, orhigher. In other embodiments, a variant CD80 decreases IFN-gammaexpression (i.e. protein expression) relative to a wild-type orunmodified CD80 control by at least: 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or higher. In some embodiments, the wild-type CD80control is murine CD80, such as would typically be used for a variantCD80 altered in sequence from that of a wild-type murine CD80 sequence.In some embodiments, the wild-type CD80 control is human CD80, such aswould typically be used for a variant CD80 altered in sequence from thatof a wild-type human CD80 sequence such as an CD80 sequence comprisingthe sequence of amino acids of SEQ ID NO:28, SEQ ID NO:152, or SEQ IDNO:372.

V. PHARMACEUTICAL FORMULATIONS

Provided herein are compositions containing any of the variant CD80polypeptides, immunomodulatory proteins, conjugates, engineered cells orinfectious agents described herein. The pharmaceutical composition canfurther comprise a pharmaceutically acceptable excipient. For example,the pharmaceutical composition can contain one or more excipients formodifying, maintaining or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption, or penetration of thecomposition. In some aspects, a skilled artisan understands that apharmaceutical composition containing cells may differ from apharmaceutical composition containing a protein.

In some embodiments, the pharmaceutical composition is a solid, such asa powder, capsule, or tablet. For example, the components of thepharmaceutical composition can be lyophilized. In some embodiments, thesolid pharmaceutical composition is reconstituted or dissolved in aliquid prior to administration.

In some embodiments, the pharmaceutical composition is a liquid, forexample variant CD80 polypeptides dissolved in an aqueous solution (suchas physiological saline or Ringer's solution). In some embodiments, thepH of the pharmaceutical composition is between about 4.0 and about 8.5(such as between about 4.0 and about 5.0, between about 4.5 and about5.5, between about 5.0 and about 6.0, between about 5.5 and about 6.5,between about 6.0 and about 7.0, between about 6.5 and about 7.5,between about 7.0 and about 8.0, or between about 7.5 and about 8.5).

In some embodiments, the pharmaceutical composition comprises apharmaceutically-acceptable excipient, for example a filler, binder,coating, preservative, lubricant, flavoring agent, sweetening agent,coloring agent, a solvent, a buffering agent, a chelating agent, orstabilizer. Examples of pharmaceutically-acceptable fillers includecellulose, dibasic calcium phosphate, calcium carbonate,microcrystalline cellulose, sucrose, lactose, glucose, mannitol,sorbitol, maltol, pregelatinized starch, corn starch, or potato starch.Examples of pharmaceutically-acceptable binders includepolyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol,gelatin, sucrose, polyethylene glycol, methyl cellulose, or cellulose.Examples of pharmaceutically-acceptable coatings include hydroxypropylmethylcellulose (HPMC), shellac, corn protein zein, or gelatin. Examplesof pharmaceutically-acceptable disintegrants includepolyvinylpyrrolidone, carboxymethyl cellulose, or sodium starchglycolate. Examples of pharmaceutically-acceptable lubricants includepolyethylene glycol, magnesium stearate, or stearic acid. Examples ofpharmaceutically-acceptable preservatives include methyl parabens, ethylparabens, propyl paraben, benzoic acid, or sorbic acid. Examples ofpharmaceutically-acceptable sweetening agents include sucrose,saccharine, aspartame, or sorbitol. Examples ofpharmaceutically-acceptable buffering agents include carbonates,citrates, gluconates, acetates, phosphates, or tartrates.

In some embodiments, the pharmaceutical composition further comprises anagent for the controlled or sustained release of the product, such asinjectable microspheres, bio-erodible particles, polymeric compounds(polylactic acid, polyglycolic acid), beads, or liposomes.

In some embodiments, the pharmaceutical composition is sterile.Sterilization may be accomplished by filtration through sterilefiltration membranes or radiation. Where the composition is lyophilized,sterilization using this method may be conducted either prior to orfollowing lyophilization and reconstitution. The composition forparenteral administration may be stored in lyophilized form or insolution. In addition, parenteral compositions generally are placed intoa container having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

In some embodiments, provided are pharmaceutical compositions containingthe transmembrane immunomodulatory proteins, including engineered cellsexpressing such transmembrane immunomodulatory proteins. In someembodiments, the pharmaceutical compositions and formulations includeone or more optional pharmaceutically acceptable carrier or excipient.Such compositions may comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.Compositions of the present invention are preferably formulated forintravenous administration.

In some embodiments, the pharmaceutical composition contains infectiousagents containing nucleic acid sequences encoding the immunomodulatoryvariant polypeptides. In some embodiments, the pharmaceuticalcomposition contains a dose of infectious agents suitable foradministration to a subject that is suitable for treatment. In someembodiments, the pharmaceutical composition contains an infectious agentthat is a virus, at a single or multiple dosage amount, of between aboutbetween or between about 1×10⁵ and about 1×10¹² plaque-forming units(pfu), 1×10⁶ and 1×10¹⁰ pfu, or 1×10⁷ and 1×10¹⁰ pfu, each inclusive,such as at least or at least about or at about 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹ pfu or about 1×10¹⁰ pfu. In someembodiments, the pharmaceutical composition can contain a virusconcentration of from or from about 10⁵ to about 10¹⁰ pfu/mL, forexample, 5×10⁶ to 5×10⁹ or 1×10⁷ to 1×10⁹ pfu/mL, such as at least or atleast about or at about 10⁶ pfu/mL, 10⁷ pfu/mL, 10⁸ pfu/mL or 10⁹pfu/mL. In some embodiments, the pharmaceutical composition contains aninfectious agent that is a bacterium, at a single or multiple dosageamount, of between about between or between about 1×10³ and about 1×10⁹colony-forming units (cfu), 1×10⁴ and 1×10⁹ cfu, or 1×10⁵ and 1×10⁷ cfu,each inclusive, such as at least or at least about or at about 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸ or 1×10⁹ cfu. In some embodiments, thepharmaceutical composition can contain a bacterial concentration of fromor from about 10³ to about 10⁸ cfu/mL, for example, 5×10⁵ to 5×10⁷ or1×10⁶ to 1×10⁷ cfu/mL, such as at least or at least about or at about10⁵ cfu/mL, 10⁶ cfu/mL, 10⁷ cfu/mL or 10⁸ cfu/mL

Such a formulation may, for example, be in a form suitable forintravenous infusion. A pharmaceutically acceptable carrier may be apharmaceutically acceptable material, composition, or vehicle that isinvolved in carrying or transporting cells of interest from one tissue,organ, or portion of the body to another tissue, organ, or portion ofthe body. For example, the carrier may be a liquid or solid filler,diluent, excipient, solvent, or encapsulating material, or somecombination thereof. Each component of the carrier must be“pharmaceutically acceptable” in that it must be compatible with theother ingredients of the formulation. It also must be suitable forcontact with any tissue, organ, or portion of the body that it mayencounter, meaning that it must not carry a risk of toxicity,irritation, allergic response, immunogenicity, or any other complicationthat excessively outweighs its therapeutic benefits.

In some embodiments, the pharmaceutical composition is administered to asubject. Generally, dosages and routes of administration of thepharmaceutical composition are determined according to the size andcondition of the subject, according to standard pharmaceutical practice.For example, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also beused to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. The exact dosage will bedetermined in light of factors related to the subject requiringtreatment. Dosage and administration are adjusted to provide sufficientlevels of the active compound or to maintain the desired effect. Factorsthat may be taken into account include the severity of the diseasestate, the general health of the subject, the age, weight, and gender ofthe subject, time and frequency of administration, drug combination(s),reaction sensitivities, and response to therapy.

Long-acting pharmaceutical compositions may be administered every 3 to 4days, every week, or biweekly depending on the half-life and clearancerate of the particular formulation. The frequency of dosing will dependupon the pharmacokinetic parameters of the molecule in the formulationused. Typically, a composition is administered until a dosage is reachedthat achieves the desired effect. The composition may therefore beadministered as a single dose, or as multiple doses (at the same ordifferent concentrations/dosages) over time, or as a continuousinfusion. Further refinement of the appropriate dosage is routinelymade. Appropriate dosages may be ascertained through use of appropriatedose-response data. A number of biomarkers or physiological markers fortherapeutic effect can be monitored including T cell activation orproliferation, cytokine synthesis or production (e.g., production ofTNF-α, IFN-γ, IL-2), induction of various activation markers (e.g.,CD25, IL-2 receptor), inflammation, joint swelling or tenderness, serumlevel of C-reactive protein, anti-collagen antibody production, and/or Tcell-dependent antibody response(s).

In some embodiments, the pharmaceutical composition is administered to asubject through any route, including orally, transdermally, byinhalation, intravenously, intra-arterially, intramuscularly, directapplication to a wound site, application to a surgical site,intraperitoneally, by suppository, subcutaneously, intradermally,transcutaneously, by nebulization, intrapleurally, intraventricularly,intra-articularly, intraocularly, or intraspinally.

In some embodiments, the dosage of the pharmaceutical composition is asingle dose or a repeated dose. In some embodiments, the doses are givento a subject once per day, twice per day, three times per day, or fouror more times per day. In some embodiments, about 1 or more (such asabout 2 or more, about 3 or more, about 4 or more, about 5 or more,about 6 or more, or about 7 or more) doses are given in a week. In someembodiments, multiple doses are given over the course of days, weeks,months, or years. In some embodiments, a course of treatment is about 1or more doses (such as about 2 or more does, about 3 or more doses,about 4 or more doses, about 5 or more doses, about 7 or more doses,about 10 or more doses, about 15 or more doses, about 25 or more doses,about 40 or more doses, about 50 or more doses, or about 100 or moredoses).

In some embodiments, an administered dose of the pharmaceuticalcomposition is about 1 μg of protein per kg subject body mass or more(such as about 2 μg of protein per kg subject body mass or more, about 5μg of protein per kg subject body mass or more, about 10 μg of proteinper kg subject body mass or more, about 25 μg of protein per kg subjectbody mass or more, about 50 μg of protein per kg subject body mass ormore, about 100 μg of protein per kg subject body mass or more, about250 μg of protein per kg subject body mass or more, about 500 μg ofprotein per kg subject body mass or more, about 1 mg of protein per kgsubject body mass or more, about 2 mg of protein per kg subject bodymass or more, or about 5 mg of protein per kg subject body mass ormore).

In some embodiments, a therapeutic amount of a cell composition isadministered. Typically, precise amount of the compositions of thepresent invention to be administered can be determined by a physicianwith consideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising engineered cells, e.g. T cells, as described herein may beadministered at a dosage of 104 to 109 cells/kg body weight, such as 105to 106 cells/kg body weight, including all integer values within thoseranges. Engineered cell compositions, such as T cell compositions, mayalso be administered multiple times at these dosages. The cells can beadministered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al, New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particularpatient can readily be determined by one skilled in the art of medicineby monitoring the patient for signs of disease and adjusting thetreatment accordingly.

A variety of means are known for determining if administration of atherapeutic composition of the invention sufficiently modulatesimmunological activity by eliminating, sequestering, or inactivatingimmune cells mediating or capable of mediating an undesired immuneresponse; inducing, generating, or turning on immune cells that mediateor are capable of mediating a protective immune response; changing thephysical or functional properties of immune cells; or a combination ofthese effects. Examples of measurements of the modulation ofimmunological activity include, but are not limited to, examination ofthe presence or absence of immune cell populations (using flowcytometry, immunohistochemistry, histology, electron microscopy,polymerase chain reaction (PCR)); measurement of the functional capacityof immune cells including ability or resistance to proliferate or dividein response to a signal (such as using T-cell proliferation assays andpepscan analysis based on 3H-thymidine incorporation followingstimulation with anti-CD3 antibody, anti-T-cell receptor antibody,anti-CD28 antibody, calcium ionophores, PMA (phorbol 12-myristate13-acetate) antigen presenting cells loaded with a peptide or proteinantigen; B cell proliferation assays); measurement of the ability tokill or lyse other cells (such as cytotoxic T cell assays); measurementsof the cytokines, chemokines, cell surface molecules, antibodies andother products of the cells (e.g., by flow cytometry, enzyme-linkedimmunosorbent assays, Western blot analysis, protein microarrayanalysis, immunoprecipitation analysis); measurement of biochemicalmarkers of activation of immune cells or signaling pathways withinimmune cells (e.g., Western blot and immunoprecipitation analysis oftyrosine, serine or threonine phosphorylation, polypeptide cleavage, andformation or dissociation of protein complexes; protein array analysis;DNA transcriptional, profiling using DNA arrays or subtractivehybridization); measurements of cell death by apoptosis, necrosis, orother mechanisms (e.g., annexin V staining, TUNEL assays, gelelectrophoresis to measure DNA laddering, histology; fluorogenic caspaseassays, Western blot analysis of caspase substrates); measurement of thegenes, proteins, and other molecules produced by immune cells (e.g.,Northern blot analysis, polymerase chain reaction, DNA microarrays,protein microarrays, 2-dimensional gel electrophoresis, Western blotanalysis, enzyme linked immunosorbent assays, flow cytometry); andmeasurement of clinical symptoms or outcomes such as improvement ofautoimmune, neurodegenerative, and other diseases involvingself-proteins or self-polypeptides (clinical scores, requirements foruse of additional therapies, functional status, imaging studies) forexample, by measuring relapse rate or disease severity (using clinicalscores known to the ordinarily skilled artisan) in the case of multiplesclerosis, measuring blood glucose in the case of type I diabetes, orjoint inflammation in the case of rheumatoid arthritis.

VI. ARTICLES OF MANUFACTURE AND KITS

Also provided herein are articles of manufacture comprising thepharmaceutical compositions described herein in suitable packaging.Suitable packaging for compositions (such as ophthalmic compositions)described herein are known in the art, and include, for example, vials(such as sealed vials), vessels, ampules, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Thesearticles of manufacture may further be sterilized and/or sealed.

Further provided are kits comprising the pharmaceutical compositions (orarticles of manufacture) described herein, which may further compriseinstruction(s) on methods of using the composition, such as usesdescribed herein. The kits described herein may also include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for performing any methods described herein.

VII. THERAPEUTIC APPLICATIONS

The pharmaceutical compositions described herein (includingpharmaceutical composition comprising the variant CD80 polypeptides, theimmunomodulatory proteins, the conjugates, and the engineered cellsdescribed herein) can be used in a variety of therapeutic applications,such as the treatment of a disease. For example, in some embodiments thepharmaceutical composition is used to treat inflammatory or autoimmunedisorders, cancer, organ transplantation, viral infections, and/orbacterial infections in a mammal. The pharmaceutical composition canmodulate (e.g. increase or decrease) an immune response to treat thedisease. In some embodiments, the provided methods are applicable totherapeutic administration of variant CD80 polypeptides, theimmunomodulatory proteins, the conjugates, the engineered cells andinfectious agents described herein. It is within the level of a skilledartisan, in view of the provided disclosure, to choose a format for theindication depending on the type of modulation of the immune response,e.g. increase or decrease that is desired.

In some embodiments, a pharmaceutical composition provided herein thatstimulates the immune response is administered, which can be useful, forexample, in the treatment of cancer, viral infections, or bacterialinfections. In some embodiments, the pharmaceutical composition containsa variant CD80 polypeptide in a format that exhibits agonist activity ofits cognate binding partner CD28 and/or that stimulates or initiatescostimulatory signaling via CD28. Exemplary formats of a CD80polypeptide for use in connection with such therapeutic applicationsinclude, for example, an immunomodulatory protein or “stack” of avariant CD80 polypeptide and an IgSF domain or variant thereof thatbinds to a tumor antigen (e.g. Nkp30 or affinity-modified variant) (alsocalled a “tumor-localizing IgSF domain), a conjugate containing avariant CD80 polypeptide linked to a tumor-targeting moiety (also calleda tumor-localizing moiety), an engineered cell expressing atransmembrane immunomodulatory protein, or an infectious agentcomprising a nucleic acid molecule encoding a transmembraneimmunomodulatory protein, such as for expression of the transmembraneimmunomodulatory protein in an infected cell (e.g. tumor cell or APC,e.g. dendritic cell).

In some embodiments, the pharmaceutical composition can be used toinhibit growth of mammalian cancer cells (such as human cancer cells). Amethod of treating cancer can include administering an effective amountof any of the pharmaceutical compositions described herein to a subjectwith cancer. The effective amount of the pharmaceutical composition canbe administered to inhibit, halt, or reverse progression of cancers.

The cancers that can be treated by the pharmaceutical compositions andthe treatment methods described herein include, but are not limited to,melanoma, bladder cancer, hematological malignancies (leukemia,lymphoma, myeloma), liver cancer, brain cancer, renal cancer, breastcancer, pancreatic cancer (adenocarcinoma), colorectal cancer, lungcancer (small cell lung cancer and non-small-cell lung cancer), spleencancer, cancer of the thymus or blood cells (i.e., leukemia), prostatecancer, testicular cancer, ovarian cancer, uterine cancer, gastriccarcinoma, a musculoskeletal cancer, a head and neck cancer, agastrointestinal cancer, a germ cell cancer, or an endocrine andneuroendocrine cancer. In some embodiments, the cancer is Ewing'ssarcoma. In some embodiments, the cancer is selected from melanoma, lungcancer, bladder cancer, and a hematological malignancy. In someembodiments, the cancer is a lymphoma, lymphoid leukemia, myeloidleukemia, cervical cancer, neuroblastoma, or multiple myeloma.

Human cancer cells can be treated in vivo, or ex vivo. In ex vivotreatment of a human patient, tissue or fluids containing cancer cellsare treated outside the body and then the tissue or fluids arereintroduced back into the patient. In some embodiments, the cancer istreated in a human patient in vivo by administration of the therapeuticcomposition into the patient.

In some embodiments, the pharmaceutical composition is administered as amonotherapy (i.e., as a single agent) or as a combination therapy (i.e.,in combination with one or more additional anticancer agents, such as achemotherapeutic drug, a cancer vaccine, or an immune checkpointinhibitor. In some embodiments, the pharmaceutical composition can alsobe administered with radiation therapy.

In some embodiments, the pharmaceutical composition suppresses an immuneresponse, which can be useful in the treatment of inflammatory orautoimmune disorders, or organ transplantation. In some embodiments, thepharmaceutical composition contains a variant CD80 polypeptide in aformat that exhibits antagonist activity of its cognate binding partnerCD28 and/or that blocks or inhibits costimulatory signaling via CD28.Exemplary formats of CD80 polypeptide for use in connection with suchtherapeutic applications include, for example, a variant CD80polypeptide that is soluble (e.g. variant CD80-Fc fusion protein), animmunomodulatory protein or “stack” of a variant CD80 polypeptide andanother IgSF domain, including soluble forms thereof that are Fcfusions, an engineered cell expressing a secretable immunomodulatoryprotein, or an infectious agent comprising a nucleic acid moleculeencoding a secretable immunomodulatory protein, such as for expressionand secretion of the secretable immunomodulatory protein in an infectedcell (e.g. tumor cell or APC, e.g. dendritic cell).

In some embodiments, the inflammatory or autoimmune disorder isAntineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, avasculitis, an autoimmune skin disease, transplantation, a Rheumaticdisease, an inflammatory gastrointestinal disease, an inflammatory eyedisease, an inflammatory neurological disease, an inflammatory pulmonarydisease, an inflammatory endocrine disease, or an autoimmunehematological disease.

In some embodiments, the inflammatory and autoimmune disorders that canbe treated by the pharmaceutical composition described herein isAddison's Disease, allergies, alopecia areata, Alzheimer's,anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis,ankylosing spondylitis, antiphospholipid syndrome (Hughes Syndrome),asthma, atherosclerosis, rheumatoid arthritis, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmunelymphoproliferative syndrome, autoimmune myocarditis, autoimmuneoophoritis, autoimmune orchitis, azoospermia, Behcet's Disease, Berger'sDisease, bullous pemphigoid, cardiomyopathy, cardiovascular disease,celiac Sprue/coeliac disease, chronic fatigue immune dysfunctionsyndrome (CFIDS), chronic idiopathic polyneuritis, chronic inflammatorydemyelinating, polyradicalneuropathy (CIPD), chronic relapsingpolyneuropathy (Guillain-Barré syndrome), Churg-Strauss Syndrome (CSS),cicatricial pemphigoid, cold agglutinin disease (CAD), COPD (chronicobstructive pulmonary disease), CREST syndrome, Crohn's disease,dermatitis, herpetiformus, dermatomyositis, diabetes, discoid lupus,eczema, epidermolysis bullosa acquisita, essential mixedcryoglobulinemia, Evan's Syndrome, exopthalmos, fibromyalgia,Goodpasture's Syndrome, Graves' Disease, Hashimoto's thyroiditis,idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura(ITP), IgA nephropathy, immunoproliferative disease or disorder,inflammatory bowel disease (IBD), interstitial lung disease, juvenilearthritis, juvenile idiopathic arthritis (JIA), Kawasaki's Disease,Lambert-Eaton Myasthenic Syndrome, lichen planus, lupus nephritis,lymphoscytic lypophisitis, Ménière's Disease, Miller Fish Syndrome/acutedisseminated encephalomyeloradiculopathy, mixed connective tissuedisease, multiple sclerosis (MS), muscular rheumatism, myalgicencephalomyelitis (ME), myasthenia gravis, ocular inflammation,pemphigus foliaceus, pemphigus vulgaris, pernicious anaemia,polyarteritis nodosa, polychondritis, polyglandular syndromes(Whitaker's syndrome), polymyalgia rheumatica, polymyositis, primaryagammaglobulinemia, primary biliary cirrhosis/autoimmune cholangiopathy,psoriasis, psoriatic arthritis, Raynaud's Phenomenon, Reiter'sSyndrome/reactive arthritis, restenosis, rheumatic fever, rheumaticdisease, sarcoidosis, Schmidt's syndrome, scleroderma, Sjörgen'sSyndrome, stiff-man syndrome, systemic lupus erythematosus (SLE),systemic scleroderma, Takayasu arteritis, temporal arteritis/giant cellarteritis, thyroiditis, Type 1 diabetes, ulcerative colitis, uveitis,vasculitis, vitiligo, interstitial bowel disease or Wegener'sGranulomatosis. In some embodiments, the inflammatory or autoimmunedisorder is selected from interstitial bowel disease, transplant,Crohn's disease, ulcerative colitis, multiple sclerosis, asthma,rheumatoid arthritis, and psoriasis.

In some embodiments, the pharmaceutical composition is administered tomodulate an autoimmune condition. For example, suppressing an immuneresponse can be beneficial in methods for inhibiting rejection of atissue, cell, or organ transplant from a donor by a recipient.Accordingly, in some embodiments, the pharmaceutical compositionsdescribed herein are used to limit or prevent graft-related ortransplant related diseases or disorders, such as graft versus hostdisease (GVHD). In some embodiments, the pharmaceutical compositions areused to suppress autoimmune rejection of transplanted or grafted bonemarrow, organs, skin, muscle, neurons, islets, or parenchymal cells.

Pharmaceutical compositions comprising engineered cells and the methodsdescribed herein can be used in adoptive cell transfer applications. Insome embodiments, cell compositions comprising engineered cells can beused in associated methods to, for example, modulate immunologicalactivity in an immunotherapy approach to the treatment of, for example,a mammalian cancer or, in other embodiments the treatment of autoimmunedisorders. The methods employed generally comprise a method ofcontacting a TIP of the present invention with a mammalian cell underconditions that are permissive to specific binding of the affinitymodified IgSF domain and modulation of the immunological activity of themammalian cell. In some embodiments, immune cells (such as tumorinfiltrating lymphocytes (TILs), T-cells (including CD8+ or CD4+T-cells), or APCs) are engineered to express as a membrane proteinand/or as a soluble variant CD80 polypeptide, immunomodulatory protein,or conjugate as described herein. The engineered cells can then becontact a mammalian cell, such as an APC, a second lymphocyte or tumorcell in which modulation of immunological activity is desired and underconditions that are permissive of specific binding of the affinitymodified IgSF domain to a counter-structure on the mammalian cell suchthat immunological activity can be modulated in the mammalian cell.Cells can be contacted in vivo or ex vivo.

In some embodiments, the engineered cells are autologous cells. In otherembodiments, the cells are allogeneic. In some embodiments, the cellsare autologous engineered cells reinfused into the mammal from which itwas isolated. In some embodiments, the cells are allogenic engineeredcells infused into the mammal. In some embodiments, the cells areharvested from a patient's blood or tumor, engineered to express apolypeptide (such as the variant CD80 polypeptide, immunomodulatoryprotein, or conjugate as described herein), expanded in an in vitroculture system (for example, by stimulating the cells), and reinfusedinto the patient to mediate tumor destruction. In some embodiments, themethods is conducted by adoptive cell transfer wherein cells expressingthe TIP (e.g., a T-cell) are infused back into the patient. In someembodiments, the therapeutic compositions and methods of the inventionare used in the treatment of a mammalian patient of cancers such aslymphoma, lymphoid leukemia, myeloid leukemia, cervical cancer,neuroblastoma, or multiple myeloma.

VIII. EXEMPLARY EMBODIMENTS

Among the provided embodiments are:

1. A variant CD80 polypeptide, comprising an IgV domain or a specificbinding fragment thereof, an IgC domain or a specific binding fragmentthereof, or both, wherein the variant CD80 polypeptide comprises one ormore amino acid modifications in an unmodified CD80 or specific bindingfragment thereof, corresponding to position(s) 4, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 20, 21, 22, 24, 25, 27, 28, 29, 30, 31, 33, 36, 37, 38,40, 41, 42, 43, 44, 47, 48, 50, 52, 53, 54, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 72, 74, 76, 77, 80, 81, 83, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 99, 102, 103, 104, 107, 108, 109, 110, 114,115, 116, 117, 118, 120, 121, 122, 126, 127, 128, 129, 130, 133, 137,140, 142, 143, 144, 148, 149, 152, 154, 160, 162, 164, 168, 169, 174,175, 177, 178, 183, 185, 188, 190, 192, 193, or 199 with reference tonumbering of SEQ ID NO: 28.

2. The variant CD80 polypeptide of embodiment 1, wherein the one or moreamino acid modifications comprises one or more amino acid substitution,insertion or deletion.

3. The variant CD80 polypeptide of embodiment 1 or embodiment 2, whereinthe unmodified CD80 is a mammalian CD80.

4. The variant CD80 polypeptide of any of embodiments 1-3, wherein theCD80 is a human CD80.

5. The variant CD80 polypeptide of any of embodiments 1-4, wherein thevariant CD80 polypeptide comprises:

-   -   the IgV domain or a specific binding fragment thereof; and    -   the IgC domain or a specific binding fragment thereof.

6. The variant CD80 polypeptide of any of embodiments 1-5, wherein theunmodified CD80 comprises (i) the sequence of amino acids set forth inSEQ ID NO:28, (ii) a sequence of amino acids that has at least 95%sequence identity to SEQ ID NO:28; or (iii) is a portion thereofcomprising an IgV domain or IgC domain or specific binding fragmentsthereof.

7. The variant CD80 polypeptide of any of embodiments 1-6, wherein:

the specific binding fragment of the IgV domain or the IgC domain has alength of at least 50, 60, 70, 80, 90, 100, 110 or more amino acids;

the specific binding fragment of the IgV domain comprises a length thatis at least 80% of the length of the IgV domain set forth as amino acids35-135 of SEQ ID NO:1; or

the specific binding fragment of the IgC domain comprises a length thatis at least 80% of the length of the IgC domain set forth as amino acids145-230 of SEQ ID NO:1.

8. The variant CD80 polypeptide of any of embodiments 1-7, wherein thevariant CD80 polypeptide comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications,optionally amino acid substitutions.

9. The variant CD80 polypeptide of any of embodiments 1-8, wherein thevariant CD80 polypeptide comprises a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 28, or a specificbinding fragment thereof.

10. The variant CD80 polypeptide of any of embodiments 1-9, wherein thevariant CD80 exhibits altered binding specificity to the ectodomain ofCD28, PD-L1, or CTLA-4 compared to the unmodified CD80.

11. The variant CD80 polypeptide of embodiment 9, wherein the alteredbinding is altered binding affinity and/or altered binding selectivity.

12. The variant CD80 polypeptide of any one of embodiments 1-11, whereinthe one or more amino acid substitution is V4M, K9E, E10R, V11S, A12G,A12T, A12V, T13N, L14A, S15V, S15F, C16S, C16G, C16L, G17W, H18L, H18R,H18Y, V20L, S21P, V22A, E24G, L25P, Q27R, T28A, T28S, R29C, R29D, R29H,R29V, I30V, Y31F, Y31H, Y31L, Q33H, K36E, K36G, K37E, K37Q, M38I, M38L,M38T, M38V, L40M, T41A, T41G, T41D, T41I, M42T, M43I, M43Q, M43R, M43V,S44P, M47T, N48D, N48I, W50G, E52G, Y53C, K54M, F59L, F59S, D60V, I61N,T62S, N63S, N64S, L65H, S66H, I67F, I67T, V68A, V68M, I69T, L70Q, L70P,L70R, L72P, P74L, D76G, E77G, E77K, Y80N, E81A, E81R, E81V, V83A, V83I,L85I, L85R, K86E, Y87N, E88D, E88G, K89E, K89N, K89R, D90K, D90L, D90N,A91E, A91G, A91S, A91T, F92L, F92N, F92P, F92Y, K93I, K93E, K93Q, K93R,K93V, R94G, R94L, R94F, E95K, H96R, L97R, E99D, E99G, L102S, S103L,S103P, V104A, V104L, D107N, F108L, P109S, P109H, T110A, S114T, D115G,F116S, F116L, E117V, E117G, I118V, I118A, I118T, T120S, S121P, N122S,I126L, I126V, I127T, C128Y, C128R, S129L, S129P, T130A, G133D, P137L,S140T, L142S, E143G, N144D, N144S, L148S, N149D, N149S, N152T, T154I,T154A, E160G, E162G, Y164H, S168G, K169E, K169I, K169S, M174T, M174V,T175A, N177S, H178R, L183H, K185E, H188D, H188Q, R190S, N192D, Q193L,T199S, or a conservative amino acid substitution thereof.

13. The variant CD80 polypeptide of any one of embodiments 1-12, whereinthe one or more amino acid substitution is V4M/L70Q/A91G/T120S/T130A,A12T/H18L/M43V/F59L/E77K/P109S/I118T,A12V/S15F/Y31H/T41G/T130A/P137L/N152T, V20L/L70Q/A91S/T120S/T130A,V22A/L70Q/S121P, E24G/L25P/L70Q/T120S, T28S/L70Q/A91G/E95K/T120S/T130A,E24G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/H96R,R29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S,R29H/E52G/L70R/E88G/A91G/T130A, R29H/E52G/T120S/T130A,R29V/Y31F/K36G/M38L/M43Q/E81R/V83I/L85I/K89R/D90L/A91E/F92N/K93Q/R94G,R29V/M43Q/E81R/L85I/K89R/D90L/A91E/F92N/K93Q/R94G,Y31H/T41G/L70Q/A91G/T120S/T130A, K36G, K36G/K37Q/M38I/L40M,K36G/K37Q/M38I/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/E99G/T130A/N149S,K36E/I67T/L70Q/A91G/T120S/T130A/N152T, K37E/F59S/L70Q/A91G/T120S/T130A,M38T/L70Q/E77G/A91G/T120S/T130A/N152T,M38V/T41D/M43I/W50G/D76GN83A/K89E/T120S/T130A, T41I/A91G,S44P/L70Q/A91G/T130A, E52G/L70Q/A91G/T120S/T130A, K54M/A91G/T120S,D60V/A91G/T120S/T130A, N63S/L70Q/A91G/T120S/T130A,S66H/D90G/T110A/F116L, I67F/L70R/E88G/A91G/T120S/T130A,I67T/L70Q/A91G/T120S, V68A/T110A, V68M/L70P/L72P/K86E,L70Q/A91G/T110A/T120S/T130A, L70Q/E81A/A91G/T120S/I127T/T130A,L70Q/Y87N/A91G/T130A, L70Q/A91G, L70Q/A91G/E117G/T120S/T130A,L70Q/A91G/T120S/T130A, L70Q/A91G/T130A, L70Q/A91G/I118A/T20S/T130A,L70R/A91G/T120S/T130A, E88D/K89R/D90K/A91G/F92Y/K93R, K89E/T130A,K89R/D90K/A91G/F92Y/K93R, K89R/D90K/A91G/F92Y/K93R/N122S/N177S,E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S A91G, A91G/F92L/F108L/T120S,A91G/L102S, A91G/S103P, A91G/T120S/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/M174T,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/H188D,H18R/R29D/Y31L/Q33H/K36G/K37E/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/E143G/K169E/M174V/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/F108L/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/C128Y/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/T130A/K169E,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93I/R94L/L97R/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93I/R94L/L97R/T130A/L148S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/I61N/E81V/L85R/K89N/A91T/F92P/K93V/R94F/V104A/T120S/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/F92P/K93V/R94F/I118V/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/T175A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/F116S/T130A/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/L142S/H188D,C16S/H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T110A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/A91G/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/D76G/A91G/S103L/T120S/I127T/T130A, Q33del/Y53C/L85R/K89N/A91T/F92P/K93V/R94L/T120S/I127T/T130A/K169E,T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T120S/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/S129L/H188D,K9E/E10R/V11S/A12G/T13N/K14A/S15V/C16L/G17W/H18Y/Y53C/L70Q/D90G/T130A/N149D/N152T/H188D,H18L/R29D/Y31L/Q33H/K36G/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,K89E/K93E/T130A,S21P/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N48I/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/P109H/I126L/K169I,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/P74L/Y80N/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R,S21P/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/P74L/Y80N/E81V/L85R/K89N/D90N/A91T/F92P/K93V/R94L/T130A/N149S/E162G,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/R190S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/P74L/Y80N/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/R190S,C16GN22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/D76G/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169I/H178R/N192D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/E117V/I118T/N149S/S168G/H188Q,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N64S/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118T/T130A/N149S/K169I,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/D115G/I118T/T130A/G133D/N149S,S129P, A91G/S129P, I69T/L70Q/A91G/T120S, Y31H/S129P,T28A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/V104L/T130A/N149S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/N149S/H188Q,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/N149S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/T154I,A12G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/T130A/L183H,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I/Q193L,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/T130A/N149S/K169I,I118T/C128R, Q27R/R29C/M42T/S129P/E160G, S129P/T154A,S21P/L70Q/D90G/T120S/T130A, L70Q/A91G/N144D,L70Q/A91G/I118A/T120S/T130A/K169E,V4M/L70Q/A91G/I118V/T120S/T130A/K169E,L70Q/A91G/I118V/T120S/T130A/K169E, L70Q/A91G/I118V/T120S/T130A,V20L/L70Q/A91S/I118V/T120S/T130A, L70Q/A91G/E117G/I118V/T120S/T130A,A91G/I118V/T120S/T130A, L70R/A91G/I118V/T120S/T130A/T199S,L70Q/E81A/A91G/I118V/T120S/I127T/T130A,T28S/L70Q/A91G/E95K/I118V/T120S/I126V/T130A/K169E,N63S/L70Q/A91G/S114T/I118V/T120S/T130A,K36E/I67T/L70Q/A91G/I118V/T120S/T130A/N152T,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E,K37E/F59S/L70Q/A91G/I118V/T120S/T130A/K185E,D60V/A91G/I118V/T120S/T130AK169E, K54M/L70Q/A91G/Y164H,M38T/L70Q/E77G/A91G/I118V/T120S/T130A/N152T,Y31H/T41G/M43L/L70Q/A91G/I118V/T120S/I126V/T130A,Y31H/T41G/M43L/L70Q/A91G/I118V/T120S/I126V/T130A, L65H/D90G/T110A/F116L,R29H/E52G/D90N/I118V/T120S/T130A, I67T/L70Q/A91G/I118V/T120S,L70Q/A91G/T110A/I118V/T120S/T130A,M38V/T41D/M43I/W50G/D76GN83A/K89E/I118V/T120S/I126V/T130A,A12V/S15F/Y31H/M38L/T41G/M43L/D90N/T130A/P137L/N149D/N152T,I67F/L70R/E88G/A91G/I118V/T120S/T130A,E24G/L25P/L70Q/A91G/I118V/T120S/N152T,E24G/L25P/L70Q/A91G/I118V/T120S/N152T, A91G/F92L/F108L/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S,K36G/K37Q/M38I/L40M/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/E99G/T130A/N149S,K36G/L40M,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/M174T,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N48D/F59L/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,H18R/R29D/Y31L/Q33H/K36G/K37E/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/E143G/K169E/M174V/H188D,R29D/I30V/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/F108L/I118V/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/N149D/K169E/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/C128Y/T130A/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/E99D/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/I61N/E81V/L85R/K89N/A91T/F92P/K93V/R94F/V104A/I118V/T120S/I126V/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118V/T120S/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118V/T120S/T130AR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E/T175A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/L142S/H188D,C16S/H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T110A/I118V/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/A91G/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/L70Q/D76G/A91G/S103L/I118V/T120S/I127T/T130A,Y53C/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,Y53C/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/K169E,T62S/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/T130A/K169E,Y53C/L70Q/D90G/T130A/N149D/N152T/H188D,Y53C/L70Q/D90G/T130A/N149D/N152T/H188D,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,orH18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S.

14. The variant CD80 polypeptide of any of embodiments 1-13, comprisingthe sequence of amino acids set forth in any of SEQ ID NOS: 55-108,280-346, 414-475 or a specific binding fragment thereof, or a sequenceof amino acids that exhibits at least 95% sequence identity to any ofSEQ ID NOS:55-108, 280-346, 414-475 or a specific binding fragmentthereof and that contains the one or more of the amino acidsubstitutions.

15. The variant CD80 polypeptide of any of embodiments 1-14, wherein thevariant CD80 polypeptide comprises the IgV domain or a specific bindingfragment thereof.

16. The variant CD80 polypeptide of any of embodiments 1-15, wherein theIgV domain or specific fragment thereof is the only CD80 portion of thevariant CD80 polypeptide.

17. The variant CD80 polypeptide of any of embodiments 1-14, wherein theIgC domain or specific fragment thereof is the only CD80 portion of thevariant CD80 polypeptide.

18. The variant CD80 polypeptide of any of embodiments 1-17, comprisingthe sequence of amino acids set forth in any of SEQ ID NOS: 153-195,347, 373-386, 476-477 or a specific binding fragment thereof, a sequenceof amino acids that exhibits at least 95% sequence identity to any ofSEQ ID NOS: 153-195, 347, 373-386, 476-477 or a specific bindingfragment thereof and that contains the one or more of the amino acidsubstitutions.

19. The variant CD80 polypeptide of any of embodiments 1-17, wherein thevariant CD80 polypeptide specifically binds to the ectodomain of CD28,PD-L1, or CTLA-4 with increased affinity compared to the unmodified CD80polypeptide.

20. The variant CD80 polypeptide of any of embodiments 1-19, wherein thevariant polypeptide specifically binds to the ectodomain of CD28, PD-L1or CTLA-4 with increased selectivity compared to the binding of theunmodified CD80 for the ectodomain.

21. The variant CD80 polypeptide of embodiment 20, wherein the increasedselectivity comprises a greater ratio for one cognate binding partnerselected from among CD28, PD-L1 and CTLA-4 versus another of the cognatebinding partner compared to the ratio of binding of the unmodified CD80polypeptide for the one cognate binding partner versus the another ofthe cognate binding partner.

22. The variant CD80 polypeptide of embodiment 19 or embodiment 20,wherein the variant polypeptide specifically binds to the ectodomain ofCD28 with increased selectivity compared to the binding of theunmodified CD80 for the ectodomain of CD28.

23. The variant CD80 polypeptide of embodiment 22, wherein the increasedselectivity comprises a greater ratio for binding CD28 versus PD-L1 orCTLA-4 compared to the ratio of binding of the unmodified CD80polypeptide for CD28 versus PD-L1 or CTLA-4.

24. The variant CD80 polypeptide of embodiment 21 or embodiment 23,wherein the ratio is greater by at least or at least about 1.5-fold,2.0-fold, 3.0-fold, 4.0-fold. 5-fold, 10-fold, 15-fold, 20-fold,30-fold, 40-fold, 50-fold or more.

25. The variant CD80 polypeptide of any of embodiments 1-24, wherein thevariant CD80 polypeptide specifically binds to the ectodomain of CD28with increased affinity compared to the unmodified CD80 polypeptide.

26. The variant CD80 polypeptide of embodiment 19 or embodiment 25,wherein the increased affinity to the ectodomain is increased more than1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-foldcompared to the binding affinity of the unmodified CD80 for theectodomain.

27. The variant CD80 polypeptide of any of embodiments 1-26, wherein theone or more amino acid substitutions corresponds to position(s) 12, 18,20, 29, 31, 36, 40, 41, 43, 52, 59, 60, 63, 67, 70, 77, 81, 87, 88, 89,90, 91, 92, 93, 107, 109, 114, 117, 118, 120, 122, 127, 130, 144, 169,177 or 199 with reference to numbering of SEQ ID NO: 28.

28. The variant CD80 polypeptide of any of embodiments 1-27, wherein theone or more amino acid substitution is selected from the groupconsisting of A12T, H18L, V20L, R29H, Y31H, K36G, T41G, T41I, M43V,E52G, F59L, D60V, N63S, I67T, L70Q, L70R, E77K, E81A, Y87N, E88D, E88G,K89E, K89R, D90K, D90N, L40M, A91G, A91S, F92Y, K93R, D107N, P109S,S114T, E117G, I118A, I118T, I118V, T120S, I127T, T130A, N144D, K169E,N177S, and T199S and conservative amino acid substitutions thereof.

29. The variant CD80 polypeptide of any of embodiments 1-28, wherein theone or more amino acid substitution isA12T/H18L/M43V/F59L/E77K/P109S/I118T, V20L/L70Q/A91S/T120S/T130A,V20L/L70Q/A91S/I118V/T120S/T130A,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,T41I/A91G, E52G/L70/A91G/T120S/T130A,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E, D60V/A91G/T120S/T130A,D60V/A91G/I118V/T120S/T130A/K169E, N63S/L70Q/A91G/T120S/T130A,N63S/L70Q/A91G/S114T/I118V/T120S/T130A, I67T/L70Q/A91G/T120S,I67T/L70Q/A91G/I118V/T120S, L70Q/E81A/A91G/T120S/I127T/T130A,L70Q/E81A/A91G/I118V/T120S/I127T/T130A, L70Q/Y87N/A91G/T130A, L70Q/A91G,L70Q/A91GN144D, L70Q/A91G/E117G/T120S/T130A,L70Q/A91G/E117G/I118V/T120S/T130A, L70Q/A91G/I118A/T120S/T130A,L70Q/A91G/I118A/T120S/T130A/K169E, L70Q/A91G/T120S/T130A,L70R/A91G/T120S/T130A, L70R/A91G/I118V/T120S/T130A/T199SL70Q/A91G/I118V/T120S/T130A/K169E, E88D/K89R/D90K/A91G/F92Y/K93R,K89R/D90K/A91G/F92Y/K93R, E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S, orK89R/D90K/A91G/F92Y/K93R/N122S/N177S.

30. The variant CD80 polypeptide of any of embodiments 1-29, wherein thevariant CD80 polypeptide specifically binds to the ectodomain of PD-L1with increased affinity compared to the unmodified CD80 polypeptide.

31. The variant CD80 polypeptide of embodiment 30, wherein the increasedaffinity to the ectodomain is increased more than 1.2-fold, 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold or 60-fold compared to the bindingaffinity of the unmodified CD80 for the ectodomain.

32. The variant CD80 polypeptide of any of embodiments 1-31, wherein thevariant polypeptide specifically binds to the ectodomain of PD-L1 withincreased selectivity compared to the binding of the unmodified CD80 forthe ectodomain.

33. The variant CD80 polypeptide of embodiment 32, wherein the increasedselectivity comprises a greater ratio for binding PD-L1 versus CD28 orCTLA-4 compared to the ratio of binding of the unmodified CD80polypeptide for PD-L1 versus CD28 or CTLA-4.

34. The variant CD80 polypeptide of embodiment 33, wherein the ratio isgreater by at least or at least about 1.5-fold, 2.0-fold, 3.0-fold,4.0-fold. 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-foldor more.

35. The variant CD80 polypeptide of any of embodiments 1-34, wherein theone or more amino acid substitutions corresponds to position(s) 12, 18,29, 31, 33, 36, 38, 40, 41, 42, 43, 47, 48, 59, 64, 67, 68, 70, 77, 81,85, 87, 88, 89, 90, 91, 92, 93, 94, 97, 104, 109, 115, 117, 118, 120,122, 126, 130, 133, 140, 144, 148, 149, 168, 178, 183, 188 or 193 withreference to numbering of SEQ ID NO: 28.

36. The variant CD80 polypeptide of any of embodiments 1-35, wherein theone or more amino acid substitution is selected from the groupconsisting of A12G, A12T, H18L, S21P, V22A, T28A, R29D, R29H, Y31H,Y31L, Q33H, K36G, M38I, M38L, L40M, T41A, T41G, M42T, M43R, M43V, M47T,N48I, F59L, N64S, I67T, V68A, V68M, L70Q, E77K, E81V, L85R, Y87N, E88D,E88G, K89E, K89N, K89R, D90K, D90N, A91G, A91T, F92P, F92Y, K93V, R94F,R94L, L97R, S103L, S103P, V104L, P109H, P109S, D115G, E117V, I118T,I118V, T120S, N122S, I126L, T130A, G133D, S140T, N144S, L148S, N149S,S168G, K169I, K169S, N177S, H178R, L183H, H188Q, R190S and Q193L, andconservative amino acid substitutions thereof.

37. The variant CD80 polypeptide of any of embodiments 1-36, wherein theone or more amino acid substitution isA12T/H18L/M43V/F59L/E77K/P109S/I118T,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R, K89R/D90K/A91G/F92Y/K93R, A91G,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R, K89R/D90K/A91G/F92Y/K93R, A91G,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118V/T120S/I127T/T130A/H188D,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S/R190S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/E117V/I118T/N149S/S168G/H188Q,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N64S/E81V/L85R/K89N/A91T/F92P/K93V/R94F/I118T/T130A/N149S/K169I,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/D115G/I118T/T130A/G133D/N149S,T28A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/V104L/T130A/N149S,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/N149S/H188Q,K89E/T130A, K89E/K93E/T130A,S21P/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/N48I/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/S21P/N48I/V68A/P109H/I126L/K169I,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68M/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A,H18L/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/T130A/N149S,A12G/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/V68A/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L97R/T130A/L183H,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/S140T/N149S/K169S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I/Q193L,V22A/R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/T130A/N149S/K169I,orR29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94F/T130A/N149S/K1691.

38. The variant CD80 polypeptide of any of embodiments 1-37, wherein thevariant CD80 polypeptide specifically binds to the ectodomain of CD28and the ectodomain of PD-L1 with increased affinity compared to theunmodified CD80 polypeptide.

39. The variant CD80 polypeptide of any of embodiments 1-38, wherein theone or more amino acid substitutions corresponds to position(s) 12, 18,36, 40, 43, 59, 77, 88, 89, 90, 91, 92, 93, 94, 109, 118, 122, or 177with reference to numbering of SEQ ID NO: 28.

40. The variant CD80 polypeptide of any of embodiments 1-39, wherein theone or more amino acid substitution is selected from the groupconsisting of A12T, H18L, K36G, L40M, M43V, F59L, E77K, E88D, K89R,D90K, A91G, F92Y, K93R, P109S, I118T, N112S, N177S, and conservativeamino acid substitutions thereof.

41. The variant CD80 polypeptide of any of embodiments 1-40, wherein theone or more amino acid substitution isA12T/H18L/M43V/F59L/E77K/P109S/I118T, K36G, K36G/L40M,E88D/K89R/D90K/A91G/F92Y/K93R, K89R/D90K/A91G/F92Y/K93R,E88D/K89R/D90K/A91G/F92Y/K93R/N122S/N177S, orK89R/D90K/A91G/F92Y/K93R/N122S/N177S.

42. The variant CD80 polypeptide of any of embodiments 1-41, wherein thevariant CD80 polypeptide specifically binds to the ectodomain of CTLA-4with increased affinity compared to the unmodified CD80 polypeptide.

43. The variant CD80 polypeptide of embodiment 42, wherein the increasedaffinity to the ectodomain is increased more than 1.2-fold, 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold or 60-fold compared to the bindingaffinity of the unmodified CD80 for the ectodomain.

44. The variant CD80 polypeptide of any of embodiments 1-43, wherein thevariant polypeptide specifically binds to the ectodomain of CTLA-4 withincreased selectivity compared to the binding of the unmodified CD80 forthe ectodomain.

45. The variant CD80 polypeptide of embodiment 44, wherein the increasedselectivity comprises a greater ratio for binding CTLA-4 versus CD28 orPD-L1 compared to the ratio of binding of the unmodified CD80polypeptide for CTLA-4 versus CD28 or PD-L1.

46. The variant CD80 polypeptide of embodiment 45, wherein the ratio isgreater by at least or at least about 1.5-fold, 2.0-fold, 3.0-fold,4.0-fold. 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-foldor more.

47. The variant CD80 polypeptide of any of embodiments 1-46, wherein theone or more amino acid substitutions corresponds to position(s) 4, 29,31, 36, 40, 41, 52, 67, 68, 70, 87, 88, 89, 90, 91, 92, 93, 107, 109,110, 118, 120, 130, 144, or 169 with reference to numbering of SEQ IDNO: 28.

48. The variant CD80 polypeptide of any of embodiments 1-47, wherein theone or more amino acid substitution is selected from the groupconsisting of V4M, R29H, Y31H, K36G, L40M T41G, E52G, I67T, V68A, L70Q,Y87N, E88D, E88G, K89E, K89R, D90K, D90N, A91G, F92Y, K93R, D107N,P109S, T110A, I118V, T120S, T130A, N144D, and K169E and conservativeamino acid substitutions thereof.

49. The variant CD80 polypeptide of any of embodiments 1-48, wherein theone or more amino acid substitution is V4M/L70Q/A91G/T120S/T130A,V4M/L70Q/A91G/I118V/T120S/T130A/K169E,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,E52G/L70Q/A91G/T120S/T130A,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E, I67T/L70Q/A91G/T120S,I67T/L70Q/A91G/I118V/T120S, V68A/T110A, L70Q/A91G, L70Q/A91G/N144D,L70Q/A91G/T120S/T130A, L70Q/A91G/I118V/T120S/T130A/K169E,L70Q/A91G/T130A, K89R/D90K/A91G/F92Y/K93R,E88D/K89R/D90K/A91G/F92Y/K93R, A91G/I118V/T120S/T130A, orA91G/T120S/T130A.

50. The variant CD80 polypeptide of any of embodiments 1-49, wherein thevariant CD80 polypeptide specifically binds to the ectodomain of CD28and the ectodomain of CTLA-4 with increased affinity compared to theunmodified CD80 polypeptide.

51. The variant CD80 polypeptide of any of embodiments 1-50, wherein theone or more amino acid substitutions corresponds to position(s) 36, 40,52, 70, 88, 89, 90, 91, 92, 93, 107, 118, 120, 130, 144, or 169 of SEQID NO: 28.

52. The variant CD80 polypeptide of any of embodiments 1-51, wherein theone or more amino acid substitution is selected from the groupconsisting of K36G, L40M, E52G, L70Q, E88D, K89R, D90K, A91G, F92Y,K93R, D107N, I118V, T120S, T130A, N144D, and K169E, and conservativeamino acid substitutions thereof.

53. The variant CD80 polypeptide of any of embodiments 1-52, wherein theone or more amino acid substitution is K36G, K36G/L40M, K36G/L40M,K36G/L40M, E52G/L70Q/A91G/T120S/T130A,E52G/L70Q/A91G/D107N/I118V/T120S/T130A/K169E, L70Q/A91G,L70Q/A91G/N144D, L70Q/A91G/T120S/T130A,L70Q/A91G/I118V/T120S/T130A/K169E, E88D/K89R/D90K/A91G/F92Y/K93R, orK89R/D90K/A91G/F92Y/K93R.

54. The variant CD80 polypeptide of any of embodiments 1-53, wherein thevariant CD80 polypeptide specifically binds to the ectodomain of PD-L1and the ectodomain of CTLA-4 with increased affinity compared to theunmodified CD80 polypeptide.

55. The variant CD80 polypeptide of any of embodiments 1-54, wherein theone or more amino acid substitutions corresponds to position(s) 29, 31,36, 40, 41, 67, 70, 87, 88, 89, 90, 91, 92, 93, 109, 118, 120, 122, 130,or 178 of SEQ ID NO: 28.

56. The variant CD80 polypeptide of any of embodiments 1-55, wherein theone or more amino acid substitution is selected from the groupconsisting of R29H, Y31H, K36G, L40M, T41G, I67T, L70Q, Y87N, E88D,E88G, K89E, K89R, D90N, D90K, A91G, F92Y, K93R, P109S, I118V, T120S, andconservative amino acid substitutions thereof.

57. The variant CD80 polypeptide of any of embodiments 1-56, wherein theone or more amino acid substitution isR29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S, K36G, K36G/L40M,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S,E88D/K89R/D90K/A91G/F92Y/K93R, or K89R/D90K/A91G/F92Y/K93R.

58. The variant CD80 polypeptide of any of embodiments 1-57, wherein thevariant CD80 polypeptide specifically binds to the ectodomain of CD28,the ectodomain of PD-L1, and the ectodomain of CTLA-4 with increasedaffinity compared to the unmodified CD80 polypeptide.

59. The variant CD80 polypeptide of any of embodiments 1-58, wherein theone or more amino acid substitutions corresponds to position(s) 36, 40,88, 89, 90, 91, 92, or 93 of SEQ ID NO: 28.

60. The variant CD80 polypeptide of any of embodiments 1-59, wherein theone or more amino acid substitution is selected from the groupconsisting of K36G, L40M, E88D, K89R, D90K, A91G, F92Y, K93R, andconservative amino acid substitutions thereof.

61. The variant CD80 polypeptide of any of embodiments 1-60, wherein theone or more amino acid substitution is K36G, K36G/L40ME88D/K89R/D90K/A91G/F92Y/K93R, or K89R/D90K/A91G/F92Y/K93R.

62. The variant CD80 polypeptide of any of embodiments 1-41, wherein:

the variant CD80 polypeptide specifically binds to the ectodomain ofCD28 or the ectodomain of PD-L1 with increased affinity compared to theunmodified CD80 polypeptide, and

the variant CD80 polypeptide specifically binds to the ectodomain ofCTLA-4 with decreased affinity compared to the unmodified CD80polypeptide.

63. The variant CD80 polypeptide of any of embodiments 1-41 and 62,wherein the one or more amino acid substitutions corresponds toposition(s) 29, 31, 33, 36, 38, 41, 42, 43, 47, 63, 67, 70, 81, 85, 87,88, 89, 90, 91, 92, 93, 94, 109, 114, 118, 120, 127, 130, 144, 148, or149 with reference to numbering of SEQ ID NO: 28.

64. The variant CD80 polypeptide of any of embodiments 1-41, 62, and 63,wherein the one or more amino acid substitution is selected from thegroup consisting of R29D, R29H, Y31H, Y31L, Q33H, K36G, M38I, T41A,T41G, M42T, M43R, M47T, N63S, I67T, L70Q, E81A, E81V, L85R, Y87N, E88G,K89E, K89N, D90N, A91G, A91T, F92P, K93V, R94L, P109S, S114T, I118T,I118V, T120S, I127T, T130A, N144S, L148S, and N149S, and conservativeamino acid substitutions thereof.

65. The variant CD80 polypeptide of any of embodiments 1-41 and 62-64,wherein the one or more amino acid substitution isN63S/L70Q/A91G/T120S/T130A, N63S/L70Q/A91G/S114T/I118V/T120S/T130A orL70Q/Y87N/A91G/T120S/I127T/T130A.

66. The variant CD80 polypeptide of any of embodiments 1-41 and 62-65,wherein: the variant CD80 polypeptide specifically binds to theectodomain of CD28 with increased affinity compared to the unmodifiedCD80 polypeptide, and the variant CD80 polypeptide specifically binds tothe ectodomain of CTLA-4 with decreased affinity compared to theunmodified CD80 polypeptide.

67. The variant CD80 polypeptide of any of embodiments 1-41 and 62-66,wherein the one or more amino acid substitutions corresponds toposition(s) 63, 70, 81, 87, 91, 114, 118, 120, 127, or 130 of SEQ ID NO:28.

68. The variant CD80 polypeptide of any of embodiments 1-41, and 62-67,wherein the one or more amino acid substitution is selected from thegroup consisting of N63S, L70Q, E81A, Y87N, A91G, S114T, I118V, T120S,I127T, and T130A, and conservative amino acid substitutions thereof.

69. The variant CD80 polypeptide of any of embodiments 1-41, and 62-67,wherein the one or more amino acid substitution isR29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S,N63S/L70Q/A91G/T120S/T130A, N63S/L70Q/A91G/S114T/I118V/T120S/T130A,I67T/L70Q/A91G/T120S, I67T/L70Q/A91G/I118V/T120S, orL70Q/Y87N/A91G/T120S/I127T/T130A.

70. The variant CD80 polypeptide of any of embodiments 1-41 and 62-69,wherein:

the variant CD80 polypeptide specifically binds to the ectodomain ofPD-L1 with increased affinity compared to the unmodified CD80polypeptide, and

the variant CD80 polypeptide specifically binds to the ectodomain ofCTLA-4 with decreased affinity compared to the unmodified CD80polypeptide.

71. The variant CD80 polypeptide of any of embodiments 1-41 and 62-70,wherein the one or more amino acid substitutions corresponds toposition(s) 29, 31, 33, 36, 38, 41, 42, 43, 47, 67, 70, 81, 85, 87, 88,89, 90, 91, 92, 93, 94, 109, 118, 120, 144, 148, or 149 of SEQ ID NO:28.

72. The variant CD80 polypeptide of any of embodiments 1-41, and 62-71,wherein the one or more amino acid substitution is selected from thegroup consisting of R29D, R29H, Y31H, Y31L, Q33H, K36G, M38I, T41A,T41G, M42T, M43R, M47T, I67T, L70Q, E81V, L85R, Y87N, E88G, K89E, K89N,D90N, A91G, A91T, F92P, K93V, R94L, P109S, I118T, I118V, T120S, N144S,L148S, and N149S, and conservative amino acid substitutions thereof.

73. The variant CD80 polypeptide of any of embodiments 1-41, and 62-72,wherein the one or more amino acid substitution isR29D/Y31L/Q33H/K36G/M38I/T41A/M42T/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/L148S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/I118T/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N144S/N149S,R29D/Y31L/Q33H/K36G/M38I/T41A/M43R/M47T/E81V/L85R/K89N/A91T/F92P/K93V/R94L/N149S,R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/A91G/P109S,I67T/L70Q/A91G/I118V/T120S or I67T/L70Q/A91G/T120S.

74. The variant CD80 polypeptide of any of embodiments 1-41 and 62-73,wherein:

the variant CD80 polypeptide specifically binds to the ectodomain ofCD28 and the ectodomain of PD-L1 with increased affinity compared to theunmodified CD80 polypeptide, and

the variant CD80 polypeptide specifically binds to the ectodomain ofCTLA-4 with decreased affinity compared to the unmodified CD80polypeptide.

75. The variant CD80 polypeptide of any of embodiments 1-41 and 62-74,wherein the one or more amino acid substitutions corresponds toposition(s) of 70, 81, 87, 91, or 120 of SEQ ID NO: 28.

76. The variant CD80 polypeptide of any of embodiments 1-41, and 62-75,wherein the one or more amino acid substitution is selected from thegroup consisting of L70Q, Y87N, A91G, and T120S, and conservative aminoacid substitutions thereof.

77. The variant CD80 polypeptide of any of embodiments 10-76, whereinthe CD28 is a human CD28.

78. The variant CD80 polypeptide of any of embodiments 10-77, whereinthe PD-L1 is a human PD-L1.

79. The variant CD80 polypeptide of any of embodiments 10-78, whereinthe CTLA-4 is a human CTLA-4.

80. The variant CD80 polypeptide of any of embodiments 1-79, wherein thebinding activity is altered (increased or decreased) more than 1.2-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold compared to theunmodified CD80 polypeptide.

81. The variant CD80 polypeptide of any of embodiments 1-80 that is asoluble protein.

82. The variant CD80 polypeptide of any of embodiments 1-81 that islinked to a multimerization domain.

83. The variant CD80 polypeptide of any of embodiments 1-81, wherein thevariant CD80 polypeptide is a multimeric polypeptide, optionally adimeric polypeptide, comprising a first variant CD80 polypeptide linkedto a multimerization domain and a second variant CD80 polypeptide linkedto a multimerization domain.

84. The variant CD80 polypeptide of embodiment 83, wherein the firstvariant CD80 polypeptide and the second variant CD80 polypeptide are thesame or different.

85. The variant CD80 polypeptide of any of embodiments 82-84, whereinthe multimerization domain is an Fc domain or a variant thereof withreduced effector function.

86. The variant CD80 polypeptide of any of embodiments 1-85 that islinked to a moiety that increases biological half-life of thepolypeptide.

87. The variant CD80 polypeptide of any of embodiments 1-86 that islinked to an Fc domain or a variant thereof with reduced effectorfunction.

88. The variant CD80 polypeptide of any of embodiments 85-87, wherein:

the Fc domain is mammalian, optionally human; or

the variant Fc domain comprises one or more amino acid modificationscompared to an unmodified Fc domain that is mammalian, optionally human.

89. The variant CD80 polypeptide of any of embodiments 85-88, whereinthe Fc domain or variant thereof comprises the sequence of amino acidsset forth in SEQ ID NO:226 or SEQ ID NO:227 or a sequence of amino acidsthat exhibits at least 85% sequence identity to SEQ ID NO:226 or SEQ IDNO:227.

90. The variant CD80 polypeptide of any of embodiments 82-89, whereinthe variant CD80 polypeptide is linked indirectly via a linker.

91. The variant CD80 polypeptide of any of embodiments 1-90, that is atransmembrane immunomodulatory protein further comprising atransmembrane domain linked to the extracellular domain (ECD) orspecific binding fragment thereof of the variant CD80 polypeptide.

92. The variant CD80 polypeptide of embodiment 91, wherein thetransmembrane domain comprises the sequence of amino acids set forth asresidues 243-263 of SEQ ID NO: 1 or a functional variant thereof thatexhibits at least 85% sequence identity to residues 243-263 of SEQ IDNO: 1.

93. The variant CD80 polypeptide of embodiment 91 or embodiment 92,further comprising a cytoplasmic domain linked to the transmembranedomain.

94. The variant CD80 polypeptide of embodiment 93, wherein thecytoplasmic domain comprises the sequence of amino acids set forth asresidues 264-288 of SEQ ID NO: 1 or a functional variant thereof thatexhibits at least 85% sequence identity to residues 264-288 of SEQ IDNO: 1.

95. The variant CD80 polypeptide of any of embodiments 1-94, wherein thevariant CD80 increases IFN-gamma (interferon-gamma) expression relativeto the unmodified CD80 in an in vitro primary T-cell assay.

96. The variant CD80 polypeptide of any of embodiments 1-95, wherein thevariant CD80 decreases IFN-gamma (interferon-gamma) expression relativeto the unmodified CD80 in an in vitro primary T-cell assay.

97. The variant CD80 polypeptide of any of embodiments 1-96 that isdeglycosylated. 98. An immunomodulatory polypeptide, comprising thevariant CD80 of any of embodiments 1-97 linked to a second polypeptidecomprising an immunoglobulin superfamily (IgSF) domain.

99. The immunomodulatory polypeptide of embodiment 98, wherein the IgSFdomain is affinity modified and exhibits altered binding to one or moreof its cognate binding partner(s) compared to the unmodified orwild-type IgSF domain.

100. The immunomodulatory polypeptide of embodiment 99, wherein the IgSFdomain exhibits increased binding to one or more of its cognate bindingpartner(s) compared to the unmodified or wild-type IgSF domain.

101. The immunomodulatory polypeptide of any of embodiments 98-100,wherein the variant CD80 is a first variant CD80 and the IgSF domain ofthe second polypeptide is an IgSF domain from a second variant CD80 ofany of embodiments 1-97, wherein the first and second variant CD80 arethe same or different.

102. The immunomodulatory protein of any of embodiments 98-101, whereinthe variant CD80 polypeptide is capable of specifically binding to CD28,PD-L1, or CTLA-4, and the IgSF domain of the second polypeptide iscapable of binding to a cognate binding partner other than onespecifically bound by the variant CD80 polypeptide.

103. The immunomodulatory protein of any of embodiments 98-102, whereinthe variant CD80 polypeptide is capable of specifically binding to CD28or PD-L1, and the IgSF domain is capable of binding to a cognate bindingpartner other than one specifically bound by the variant CD80polypeptide.

104. The immunomodulatory protein of any of embodiments 98-103, whereinthe variant CD80 polypeptide is capable of specifically binding to CD28or CTLA-4, and the IgSF domain is capable of binding to a cognatebinding partner other than one specifically bound by the variant CD80polypeptide.

105. The immunomodulatory protein any of embodiments 98-103, wherein thevariant CD80 polypeptide is capable of specifically binding to CTLA-4 orPD-L1 and the IgSF domain is capable of binding to a cognate bindingpartner other than one specifically bound by the variant CD80polypeptide.

106. The immunomodulatory polypeptide of any of embodiments 98-105,wherein the IgSF domain is from a member of the B7 family.

107. The immunomodulatory polypeptide of any of embodiments 82-90,wherein the IgSF domain is a tumor-localizing moiety that binds to aligand expressed on a tumor.

108. The immunomodulatory polypeptide of embodiment 107, wherein theligand is B7H6.

109. The immunomodulatory polypeptide of embodiment 107 or embodiment108, wherein the IgSF domain is from NKp30.

110. The immunomodulatory polypeptide of any of embodiments 98-109,wherein the IgSF domain is affinity modified and exhibits increasedbinding to one or more of its cognate binding partner(s) compared to theunmodified or wild-type IgSF domain.

111. The immunomodulatory polypeptide of any of embodiments 98-110,wherein the IgSF domain is or comprises an IgV domain.

112. The immunomodulatory polypeptide of any of embodiments 98-111,wherein the variant CD80 polypeptide is or comprise an IgV domain.

113. The immunomodulatory protein of any of embodiments 98-112, whereinthe immunomodulatory protein comprises a multimerization domain linkedto one or both of the variant CD80 polypeptide and the secondpolypeptide comprising the IgSF domain.

114. The immunomodulatory protein of embodiment 113, wherein themultimerization domain is an Fc domain or a variant thereof with reducedeffector function.

115. The immunomodulatory protein of any of embodiments 98-114 that isdimeric.

116. The immunomodulatory protein of embodiment 115 that is homodimeric.

117. The immunomodulatory protein of embodiment 115 that isheterodimeric.

118. A conjugate, comprising a variant CD80 of any of embodiments 1-98or an immunomodulatory polypeptide of any of embodiments 99-117 linkedto a moiety.

119. The conjugate of embodiment 118, wherein the moiety is a targetingmoiety that specifically binds to a molecule on the surface of a cell.

120. The conjugate of embodiment 119, wherein the targeting moietyspecifically binds to a molecule on the surface of an immune cell.

121. The conjugate of embodiment 120, wherein the immune cell is anantigen presenting cell or a lymphocyte.

122. The conjugate of embodiment 119, wherein the targeting moiety is atumor-localizing moiety that binds to a molecule on the surface of atumor.

123. The conjugate of any of embodiments 118-122, wherein the moiety isa protein, a peptide, nucleic acid, small molecule or nanoparticle.

124. The conjugate of any of embodiments 118-123, wherein the moiety isan antibody or antigen-binding fragment.

125. The conjugate of any of embodiments 118-124, wherein the conjugateis divalent, tetravalent, hexavalent or octavalent.

126. A nucleic acid molecule(s) encoding the variant CD80 polypeptide ofany of embodiments 1-98 or the immunomodulatory polypeptide of any ofembodiments 99-117.

127. The nucleic acid molecule of embodiment 126 that is syntheticnucleic acid.

128. The nucleic acid molecule of embodiment 126 or embodiment 127 thatis cDNA.

129. A vector, comprising the nucleic acid molecule of any ofembodiments 125-128.

130. The vector of embodiment 129 that is an expression vector.

131. The vector of embodiments 129 or embodiment 130, wherein the vectoris a mammalian vector or a viral vector.

132. A cell comprising the vector of any of embodiments 129-131.

133. The cell of embodiment 132 that is a mammalian cell.

134. The cell of embodiment 133 that is a human cell.

135. A method of producing a variant CD80 polypeptide or animmunomodulatory protein, comprising introducing the nucleic acidmolecule of any of embodiments 125-128 or vector of any of embodiments129-131 into a host cell under conditions to express the protein in thecell.

136. The method of embodiment 135, further comprising isolating orpurifying the variant CD80 polypeptide or immunomodulatory protein fromthe cell.

137. A method of engineering a cell expressing a variant CD80polypeptide, comprising introducing a nucleic acid molecule encoding thevariant CD80 polypeptide of any of embodiments 1-98 into a host cellunder conditions in which the polypeptide is expressed in the cell.

138. An engineered cell, expressing the variant CD80 polypeptide of anyof embodiments 1-98, the immunomodulatory protein of any of embodiments99-117, the nucleic acid molecule of any of embodiments 125-128 or thevector of any of embodiments 129-131.

139. The engineered cell of embodiment 138, wherein the variant CD80polypeptide or immunomodulatory polypeptide comprises a signal peptide.

140. The engineered cell of embodiment 138 or embodiment 139, whereinthe variant CD80 polypeptide or immunomodulatory polypeptide does notcomprise a transmembrane domain and/or is not expressed on the surfaceof the cell.

141. The engineered cell of any of embodiments 138-140, wherein thevariant CD80 polypeptide or immunomodulatory polypeptide is secretedfrom the engineered cell.

142. The engineered cell of embodiment 138 or embodiment 139, whereinthe engineered cell comprises a variant CD80 polypeptide that comprisesa transmembrane domain and/or is the transmembrane immunomodulatoryprotein of any of embodiments 91-97.

143. The engineered cell of embodiment 138, embodiment 139 or embodiment142, wherein the variant CD80 polypeptide is expressed on the surface ofthe cell.

144. The engineered cell of any of embodiments 138-143, wherein the cellis an immune cell.

145. The engineered cell of embodiment 144, wherein the immune cell isan antigen presenting cell (APC) or a lymphocyte.

146. The engineered cell of any of embodiments 138-145 that is a primarycell.

147. The engineered cell of any of embodiments 138-146, wherein the cellis a mammalian cell.

148. The engineered cell of any of embodiments 138-147, wherein the cellis a human cell.

149. The engineered cell of any of embodiments 145-148, wherein thelymphocyte is a T cell.

150. The engineered cell of any one of embodiments 145-149, wherein theAPC is an artificial APC.

151. The engineered cell of any of embodiments 138-150, furthercomprising a chimeric antigen receptor (CAR) or an engineered T-cellreceptor.

152. An infectious agent, comprising a nucleic acid molecule encoding avariant CD80 polypeptide of any of embodiments 1-97 or animmunomodulatory polypeptide of any of embodiments 98-117.

153. The infectious agent of embodiment 152, wherein the encoded variantCD80 polypeptide or immunomodulatory polypeptide does not comprise atransmembrane domain and/or is not expressed on the surface of a cell inwhich it is expressed.

154. The infectious agent of embodiment 152 or embodiment 153, whereinthe encoded variant COD80 polypeptide or immunomodulatory polypeptide issecreted from a cell in which it is expressed.

155. The infectious agent of embodiment 152, wherein the encoded variantCD80 polypeptide comprises a transmembrane domain.

156. The infectious agent of embodiment 152 or embodiment 155, whereinthe encoded variant CD80 polypeptide is expressed on the surface of acell in which it is expressed.

157. The infectious agent of any of embodiments 152-156, wherein theinfectious agent is a bacterium or a virus.

158. The infectious agent of embodiment 157, wherein the virus is anoncolytic virus.

159. The infectious agent of embodiment 158, wherein the oncolytic virusis an adenoviruses, adeno-associated viruses, herpes viruses, HerpesSimplex Virus, Vesticular Stomatic virus, Reovirus, Newcastle Diseasevirus, parvovirus, measles virus, vesticular stomatitis virus (VSV),Coxsackie virus or a Vaccinia virus.

160. The infectious agent of embodiment 159, wherein the virusspecifically targets dendritic cells (DCs) and/or is dendriticcell-tropic.

161. The infectious agent of embodiment 160, wherein the virus is alentiviral vector that is pseudotyped with a modified Sindbis virusenvelope product.

162. The infectious agent of any of embodiments 152-161, furthercomprising a nucleic acid molecule encoding a further gene product thatresults in death of a target cell or that can augment or boost an immuneresponse.

163. The infectious agent of embodiment 162, wherein the further geneproduct is selected from an anticancer agent, anti-metastatic agent, anantiangiogenic agent, an immunomodulatory molecule, an immune checkpointinhibitor, an antibody, a cytokine, a growth factor, an antigen, acytotoxic gene product, a pro-apoptotic gene product, an anti-apoptoticgene product, a cell matrix degradative gene, genes for tissueregeneration or a reprogramming human somatic cells to pluripotency.

164. A pharmaceutical composition, comprising the variant CD80polypeptide of any of embodiments 1-97, the immunomodulatory protein ofany of embodiments 98-117, the conjugate of any of embodiments 118-125,the engineered cell of any of embodiments 138-151 or the infectiousagent of any of embodiments 152-163.

165. The pharmaceutical composition of embodiment 164, comprising apharmaceutically acceptable excipient.

166. The pharmaceutical composition of embodiment 164 or 165, whereinthe pharmaceutical composition is sterile.

167. An article of manufacture comprising the pharmaceutical compositionof any of embodiments 164-166 in a vial.

168. The article of manufacture of embodiment 167, wherein the vial issealed.

169. A kit comprising the pharmaceutical composition of any ofembodiments 164-166, and instructions for use.

170. A kit comprising the article of manufacture according to embodiment167 and 168, and instructions for use.

171. A method of modulating an immune response in a subject, comprisingadministering the pharmaceutical composition of any of embodiments164-166 to the subject.

172. A method of modulating an immune response in a subject, comprisingadministering the engineered cells of any of embodiments 138-151.

173. The method of embodiment 172, wherein the engineered cells areautologous to the subject.

174. The method of embodiment 172, wherein the engineered cells areallogenic to the subject.

175. The method of any of embodiments 171-174, wherein modulating theimmune response treats a disease or condition in the subject.

176. The method of any of embodiments 171-175, wherein the immuneresponse is increased.

177. The method of any of embodiments 171, 175 and 176, wherein animmunomodulatory protein or conjugate comprising a variant CD80polypeptide linked to a tumor-localizing moiety is administered to thesubject.

178. The method of embodiment 177, wherein the tumor-localizing moietyis or comprises a binding molecule that recognizes a tumor antigen.

179. The method of embodiment 178, wherein the binding moleculecomprises an antibody or an antigen-binding fragment thereof orcomprises a wild-type IgSF domain or variant thereof.

180. The method of any of embodiments 171 and 175-179, wherein apharmaceutical composition comprising the immunomodulatory protein ofany of embodiments 107-117 or the conjugate of any of embodiments118-125 is administered to the subject.

181. The method of any of embodiments 172-176, wherein an engineeredcell comprising a variant CD80 polypeptide that is a transmembraneimmunomodulatory protein is administered to the subject and/or theengineered cell of 138, 139 and 142-151 is administered to the subject.

182. The method of any of embodiments 171, 175 and 176, wherein aninfectious agent encoding a variant CD80 polypeptide that is atransmembrane immunomodulatory protein is administered to the subject,optionally under conditions in which the infectious agent infects atumor cell or immune cell and the transmembrane immunomodulatory proteinis expressed on the surface of the infected cell.

183. The method of any of embodiment 181 or embodiment 182, wherein thetransmembrane immunomodulatory protein is of any of embodiments 91-97.

184. The method of any of embodiments 171-183, wherein the disease orcondition is a tumor or cancer.

185. The method of any one of embodiments 171-184, wherein the diseaseor condition is selected from melanoma, lung cancer, bladder cancer, ahematological malignancy, liver cancer, brain cancer, renal cancer,breast cancer, pancreatic cancer, colorectal cancer, spleen cancer,prostate cancer, testicular cancer, ovarian cancer, uterine cancer,gastric carcinoma, a musculoskeletal cancer, a head and neck cancer, agastrointestinal cancer, a germ cell cancer, or an endocrine andneuroendocrine cancer.

186. The method of any of embodiments 171-175, wherein the immuneresponse is decreased.

187. The method of any of embodiments 171, 175 and 186, wherein avariant CD80 polypeptide or immunomodulatory protein that is soluble isadministered to the subject.

188. The method of embodiment 187, wherein the soluble polypeptide orimmunomodulatory protein is an Fc fusion protein.

189. The method of any of embodiments 171, 175 and 186-188, wherein apharmaceutical composition comprising a variant CD80 polypeptide of anyof embodiments 1-90 and 95-97, or the immunomodulatory protein of any ofembodiments 98-106 and 111-117 is administered to the subject.

190. The method of any of embodiments 172-175 and 186, wherein anengineered cell comprising a secretable variant CD80 polypeptide isadministered to the subject.

191. The method of any of embodiments 172-175 and 186-188, wherein anengineered cell of any of embodiments 138-141 and 144-151 isadministered to the subject.

192. The method of any of embodiments 171, 175 and 186-188, wherein aninfectious agent encoding a variant CD80 polypeptide that is asecretable immunomodulatory protein is administered to the subject,optionally under conditions in which the infectious agent infects atumor cell or immune cell and the secretable immunomodulatory protein issecreted from the infected cell.

193. The method of any of embodiments 171-175 and 186-192, wherein thedisease or condition is an inflammatory or autoimmune disease orcondition.

194. The method of any of embodiments 171-175 and 186-193, wherein thedisease or condition is an Antineutrophil cytoplasmic antibodies(ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease,transplantation, a Rheumatic disease, an inflammatory gastrointestinaldisease, an inflammatory eye disease, an inflammatory neurologicaldisease, an inflammatory pulmonary disease, an inflammatory endocrinedisease, or an autoimmune hematological disease.

195. The method of embodiment 193 or embodiment 194, wherein the diseaseor condition is selected from inflammatory bowel disease, transplant,Crohn's disease, ulcerative colitis, multiple sclerosis, asthma,rheumatoid arthritis, or psoriasis.

IX. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Generation of Mutant DNA Constructs of IgSF Domains

Example 1 describes the generation of mutant DNA constructs of humanCD80 IgSF domains for translation and expression on the surface of yeastas yeast display libraries.

A. Degenerate Libraries

Constructs were generated based on a wildtype human CD80 amino acidsequence of the extracellular domain (ECD) set forth in SEQ ID NO: 28(corresponding to residues 35-242 as set forth in UniProt Accession No.P33681) as follows:

VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTT KQEHFPDN

For libraries that target specific residues for complete or partialrandomization with degenerate codons, the DNAencoding SEQ ID NO: 28 wasordered from Integrated DNA Technologies (Coralville, Iowa) as a set ofoverlapping oligonucleotides of up to 80 base pairs (bp) in length. Togenerate a library of diverse variants of the ECD, the oligonucleotidescontained desired degenerate codons at desired amino acid positions.Degenerate codons were generated using an algorithm at the URL:rosettadesign.med.unc.edu/SwiftLib/.

In general, positions to mutate and degenerate codons were chosen fromcrystal structures of the target-ligand pairs of interest to identifyligand contact residues as well as residues that are at the proteininteraction interface. This analysis was performed using a structureviewer available at the URL:spdbv.vital-it.ch). For example, a crystalstructure for CD80 bound to CTLA-4 is publicly available at theURL:www.rcsb.org/pdb/explore/explore.do?structureId=1I8L) and a targetedlibrary was designed based on the CD80::CTLA-4 interface for selectionof improved binders to CTLA-4. However, there are no CD80 structuresavailable with ligands CD28 and PDL1, so the same library was also usedto select for binders of CD28 (binds the same region on CD80 as CTLA-4)and PDL1 (not known if PDL1 binds the same site as CTLA-4).

The next step in library design was the alignment of human, mouse, ratand monkey CD80 sequences to identify conserved residues. Based on thisanalysis, conserved target residues were mutated with degenerate codonsthat only specified conservative amino acid changes plus the wild-typeresidue. Residues that were not conserved were mutated moreaggressively, but also included the wild-type residue. Degenerate codonsthat also encoded the wild-type residue were deployed to avoid excessivemutagenesis of target protein. For the same reason, only up to 20positions were targeted for mutagenesis at a time. These residues were acombination of contact residues and non-contact interface residues.

The oligonucleotides were dissolved in sterile water, mixed in equimolarratios, heated to 95° C. for five minutes and slowly cooled to roomtemperature for annealing. ECD-specific oligonucleotide primers thatanneal to the start and end of the ECDs, respectively, were then used togenerate PCR product. ECD-specific oligonucleotides which overlap by40-50 bp with a modified version of pBYDS03 cloning vector (LifeTechnologies USA), beyond and including the BamH1 and Kpn1 cloningsites, were then used to amplify 100 ng of PCR product from the priorstep to generate a total of 5 μg of DNA. Both polymerase chain reactions(PCRs) were conducted using OneTaq 2×PCR master mix (New EnglandBiolabs, USA). The products from the second PCR were purified using aPCR purification kit (Qiagen, Germany) and resuspended in steriledeionized water.

To prepare for library insertion, a modified yeast display version ofvector pBYDS03 was digested with BamH1 and Kpn1 restriction enzymes (NewEngland Biolabs, USA) and the large vector fragment was gel-purified anddissolved in sterile, deionized water. Electroporation-ready DNA for thenext step was generated by mixing 12 μg of library DNA with 4 μg oflinearized vector in a total volume of 50 μl deionized and sterilewater. An alternative way to generate targeted libraries was to carryout site-directed mutagenesis (Multisite kit, Agilent, USA) of thetarget ECD with oligonucleotides containing degenerate codons. Thisapproach was used to generate sublibraries that only target specificstretches of target protein for mutagenesis. In these cases,sublibraries were mixed before proceeding to the selection steps. Ingeneral, library sizes were in the range of 10E7 to 10E8 clones, exceptthat sublibraries were only in the range of 10E4 to 10E5. Largelibraries and sublibraries were generated for ICOSL.

B. Random Libraries

Random libraries were also constructed to identify variants of the ECDof CD80 set forth in SEQ ID NO: 28 (containing the IgV domain,corresponding to residues 35-135 as set forth in UniProt Accession No.P33681, flanked by adjacent C-terminal residues of the wildtypesequence). DNA encoding the wild-type ECD was cloned between the BamH1and Kpn1 sites of modified yeast display vector pBYDS03 and thenreleased using the same restriction enzymes. The released DNA was thenmutagenized with the Genemorph II kit (Agilent, USA) to generate anaverage of three to five amino acid changes per library variant.Mutagenized DNA was then amplified by the two-step PCR, and furtherprocessed, as described above for targeted libraries.

Example 2 Introduction of DNA Libraries into Yeast

Example 2 describes the introduction of CD80 DNA libraries into yeast.To introduce degenerate and random library DNA into yeast,electroporation-competent cells of yeast strain BJ5464 (ATCC.org; ATCCnumber 208288) were prepared and electroporated on a Gene Pulser II(Biorad, USA) with the electroporation-ready DNA from the step aboveessentially as described (Colby, D. W. et al. 2004 Methods Enzymology388, 348-358). The only exception was that transformed cells were grownin non-inducing minimal selective SCD-Leu medium to accommodate the LEU2selective marker carried by modified plasmid pBYDS03.

Library size was determined by plating dilutions of freshly recoveredcells on SCD-Leu agar plates and then extrapolating library size fromthe number of single colonies from plating that generated at least 50colonies per plate. The remainder of the electroporated culture wasgrown to saturation and cells from this culture were subcultured intothe same medium once more to minimize the fraction of untransformedcells. To maintain library diversity, this subculturing step was carriedout using an inoculum that contained at least 10× more cells than thecalculated library size. Cells from the second saturated culture wereresuspended in fresh medium containing sterile 25% (weight/volume)glycerol to a density of 10E10/ml and frozen and stored at −80° C.(frozen library stock).

One liter of SCD-Leu media consisted of 14.7 grams of sodium citrate,4.29 grams of citric acid monohydrate, 20 grams of dextrose, 6.7 gramsof Difco brand yeast nitrogen base, and 1.6 grams yeast syntheticdrop-out media supplement without leucine. Media was filtered sterilizedbefore use, using a 0.2 μM vacuum filter device.

Library size was determined by plating dilutions of freshly recoveredcells on SCD-Leu agar plates and then extrapolating library size fromthe number of single colonies from a plating that generate at least 50colonies per plate.

To segregate plasmid from cells that contain two or more differentlibrary clones, a number of cells corresponding to 10 times the librarysize, were taken from the overnight SCD-Leu culture and subcultured1/100 into fresh SCD-Leu medium and grown overnight. Cells from thisovernight culture were resuspended in sterile 25% (weight/volume)glycerol to a density of 10E10/ml and frozen and stored at −80° C.(frozen library stock).

Example 3 Yeast Selection

Example 3 describes the selection of yeast expressing affinity modifiedvariants of CD80. A number of cells equal to at least 10 times thelibrary size were thawed from individual library stocks, suspended to0.1×10E6 cells/ml in non-inducing SCD-Leu medium, and grown overnight.The next day, a number of cells equal to 10 times the library size werecentrifuged at 2000 RPM for two minutes and resuspended to 0.5×10E6cells/ml in inducing SCDG-Leu media. One liter of the SCDG-Leu inductionmedia consists of 5.4 grams Na₂HPO₄, 8.56 grams of NaH₂PO₄*H₂O, 20 gramsgalactose, 2.0 grams dextrose, 6.7 grams Difco yeast nitrogen base, and1.6 grams of yeast synthetic drop out media supplement without leucinedissolved in water and sterilized through a 0.22 μm membrane filterdevice. The culture was grown for two days at 20° C. to induceexpression of library proteins on the yeast cell surface.

Cells were processed with magnetic beads to reduce non-binders andenrich for all variant CD80 polypeptides with the ability to bind theirexogenous recombinant counter-structure proteins. For example, yeastdisplayed targeted or random CD80 libraries were selected against eachof CD28, CTLA-4 and PD-L1 separately. This was then followed by two tothree rounds of flow cytometry sorting using exogenous counter-structureprotein staining to enrich the fraction of yeast cells that displaysimproved binders. Magnetic bead enrichment and selections by flowcytometry are essentially as described in Keith D. Miller, 1 Noah B.Pefaur, 2 and Cheryl L. Baird1 Current Protocols in Cytometry4.7.1-4.7.30, July 2008.

With CD80 libraries, target ligand proteins were sourced from R&DSystems (USA) as follows: human rCD28.Fc (i.e., recombinant CD28-Fcfusion protein), rPDL1.Fc and rCTLA4.Fc. Magnetic streptavidin beadswere obtained from New England Biolabs, USA. For biotinylation ofcounter-structure protein, biotinylation kit cat #21955, LifeTechnologies, USA, was used. For two-color, flow cytometric sorting, aBecton Dickinson FACS Aria II sorter was used. CD80 display levels weremonitored with an anti-hemagglutinin (anti-HA) antibody labeled withAlexafluor 488 (Life Technologies, USA). Ligand binding Fc fusionproteins rCD28.Fc, rCTLA4.Fc and rPDL1.Fc were detected with PEconjugated human Ig specific goat Fab (Jackson ImmunoResearch, USA).Doublet yeast were gated out using forward scatter (FSC)/side scatter(SSC) parameters, and sort gates were based upon higher ligand bindingdetected in FL4 that possessed more limited tag expression binding inFL1.

Yeast outputs from the flow cytometric sorts were assayed for higherspecific binding affinity. Sort output yeast were expanded andre-induced to express the particular IgSF affinity modified domainvariants they encode. This population then can be compared to theparental, wild-type yeast strain, or any other selected outputs, such asthe bead output yeast population, by flow cytometry.

Importantly, the MFIs of all F2 outputs described above when measuredwith the anti-HA tag antibody on FL1 did not increase and sometimes wentdown compared to wild-type strains, indicating that increased bindingwas not a function of increased expression of the selected variants onthe surface of yeast, and validated gating strategies of only selectingmid to low expressors with high ligand binding.

Selected variant CD80 ECD domains were further formatted as fusionproteins and tested for binding and functional activity as describedbelow.

Example 4 Reformatting Selection Outputs as Fe-Fusions and in VariousImmunomodulatory Protein Types

Example 4 describes reformatting of selection outputs asimmunomodulatory proteins containing an affinity modified (variant)extracellular domain (ECD) of CD80 fused to an Fc molecule (variantECD-Fc fusion molecules).

Output cells from final flow cytometric CD80 sorts were grown toterminal density in SCD-Leu medium. Plasmid DNAs from each output wereisolated using a yeast plasmid DNA isolation kit (Zymoresearch, USA).For Fc fusions, PCR primers with added restriction sites suitable forcloning into the Fc fusion vector of choice were used to batch-amplifyfrom the plasmid DNA preps the coding DNA's for the mutant target ECD's.After restriction digestion, the PCR products were ligated into anappropriate Fc fusion vector followed by chemical transformation intostrain XL1 Blue E. Coli (Agilent, USA) or NEB5alpha (New EnglandBiolabs) as directed by supplier. An example of an Fc fusion vector ispFUSE-hIgG1-Fc2 (Invivogen, USA).

Dilutions of transformation reactions were plated on LB-agar containing100 μg/ml carbenicillin (Teknova, USA) to generate single colonies. Upto 96 colonies from each transformation were then grown in 96 wellplates to saturation overnight at 37° C. in LB-broth (Teknova cat#L8112) and a small aliquot from each well was submitted for DNAsequencing of the ECD insert in order to identify the mutation(s) in allclones. Sample preparation for DNA sequencing was carried out usingprotocols provided by the service provider (Genewiz; South Plainfield,N.J.). After removal of sample for DNA sequencing, glycerol was thenadded to the remaining cultures for a final glycerol content of 25% andplates were stored at −20° C. for future use as master plates (seebelow). Alternatively, samples for DNA sequencing were generated byreplica plating from grown liquid cultures to solid agar plates using adisposable 96 well replicator (VWR, USA). These plates were incubatedovernight to generate growth patches and the plates were submitted toGenewiz as specified by Genewiz. In some instances, resequencing wasperformed to verify mutations.

After identification of clones of interest from analysis ofGenewiz-generated DNA sequencing data, clones of interest were recoveredfrom master plates and individually grown to density in 5 ml liquidLB-broth containing 100 μg/ml carbenicillin (Teknova, USA) and 2 ml ofeach culture were then used for preparation of approximately 10 μg ofminiprep plasmid DNA of each clone using a standard kit such as thePureyield kit (Promega). Identification of clones of interest generallyinvolved the following steps. First, DNA sequence data files weredownloaded from the Genewiz website. All sequences were then manuallycurated so that they start at the beginning of the ECD coding region.The curated sequences were then batch-translated using a suitableprogram available at the URL: www.ebi.ac.uk/Tools/st/emboss_transeq/.The translated sequences were then aligned using a suitable programavailable at the URL: multalin.toulouse.inra.fr/multalin/multalin.html.

Clones of interest were then identified using the following criteria: 1)identical clone occurs at least two times in the alignment and 2) amutation occurs at least two times in the alignment and preferably indistinct clones. Clones that meet at least one of these criteria wereclones that have been enriched by the sorting process due to improvedbinding.

To generate immunomodulatory proteins that were Fc fusion proteinscontaining an ECD of CD80 with at least one affinity-modified domain,the encoding nucleic acid molecule was generated to encode a proteindesigned as follows: variant (mutant) ECD followed by a linker of threealanines (AAA) followed by a human IgG1 Fc containing the mutationsR77C, N82G and V87C with reference to wild-type human IgG1 Fc set forthin SEQ ID NO: 226. Since the construct does not include any antibodylight chains that can form a covalent bond with a cysteine, the humanIgG1 Fc also contains replacement of the cysteine residues to a serineresidue at position 5 (C5S) compared to the wild-type or unmodified Fcset forth in SEQ ID NO: 226.

Example 5 Expression and Purification of Fc-Fusions

Example 5 describes the high throughput expression and purification ofFc-fusion proteins containing variant ECD CD80.

Recombinant variant Fc fusion proteins were produced with Expi293expression system (Invitrogen, USA). 4 μg of each plasmid DNA from theprevious step was added to 200 μl Opti-MEM (Invitrogen, USA) at the sametime as 10.8 μl ExpiFectamine was separately added to another 200 μlOpti-MEM. After 5 minutes, the 200 μl of plasmid DNA was mixed with the200 μl of ExpiFectamine and was further incubated for an additional 20minutes before adding this mixture to cells. Ten million Expi293 cellswere dispensed into separate wells of a sterile 10 ml, conical bottom,deep 24-well growth plate (Thomson Instrument Company, USA) in a volumeof 3.4 ml Expi293 media (Invitrogen, USA). Plates were shaken for 5 daysat 120 RPM in a mammalian cell culture incubator set to 95% humidity and8% CO₂. Following a 5-day incubation, cells were pelleted and culturesupernatants were removed.

Protein was purified from supernatants using a high throughput 96-wellProtein A purification kit using the manufacturer's protocol (Catalognumber 45202, Life Technologies, USA). Resulting elution fractions werebuffer exchanged into PBS using Zeba 96-well spin desalting plate(Catalog number 89807, Life Technologies, USA) using the manufacturer'sprotocol. Purified protein was quantitated using 280 nm absorbancemeasured by Nanodrop instrument (Thermo Fisher Scientific, USA), andprotein purity was assessed by loading 5 μg of protein on NUPAGEpre-cast, polyacrylamide gels (Life Technologies, USA) under denaturingand reducing conditions and subsequent gel electrophoresis. Proteinswere visualized in gel using standard Coomassie staining.

Example 6 Assessment of Binding and Activity of Affinity-Matured IgSFDomain-Containing Molecules

A. Binding to Cell-Expressed Counter Structures

This Example describes Fc-fusion binding studies to show specificity andaffinity of CD80 domain variant immunomodulatory proteins for cognatebinding partners.

To produce cells expressing cognate binding partners, full-lengthmammalian surface expression constructs for each of human CD28, CTLA-4and PD-L1, were designed in pcDNA3.1 expression vector (LifeTechnologies) and sourced from Genscript, USA. Binding studies werecarried out using the Expi293F transient transfection system (LifeTechnologies, USA). The number of cells needed for the experiment wasdetermined, and the appropriate 30 ml scale of transfection wasperformed using the manufacturer's suggested protocol. For each CD28,CTLA-4, PD-L1, or mock 30 ml transfection, 75 million Expi293F cellswere incubated with 30 μg expression construct DNA and 1.5 ml dilutedExpiFectamine 293 reagent for 48 hours, at which point cells wereharvested for staining.

For staining by flow cytometry, 200,000 cells of appropriate transienttransfection or negative control were plated in 96-well round bottomplates. Cells were spun down and resuspended in staining buffer (PBS(phosphate buffered saline), 1% BSA (bovine serum albumin), and 0.1%sodium azide) for 20 minutes to block non-specific binding. Afterwards,cells were centrifuged again and resuspended in staining buffercontaining 100 nM to 1 nM variant immunomodulatory protein, depending onthe experiment of each candidate variant CD80 Fc protein in 50 μl.Primary staining was performed on ice for 45 minutes, before washingcells in staining buffer twice. PE-conjugated anti-human Fc (JacksonImmunoResearch, USA) was diluted 1:150 in 50 μl staining buffer andadded to cells and incubated another 30 minutes on ice. Secondaryantibody was washed out twice, cells were fixed in 4% formaldehyde/PBS,and samples were analyzed on FACScan flow cytometer (Becton Dickinson,USA).

Mean Fluorescence Intensity (MFI) was calculated for each transfectantand negative parental line with Cell Quest Pro software (BectonDickinson, USA).

B. Bioactivity Characterization

This Example further describes Fc-fusion variant protein bioactivitycharacterization in human primary T cell in vitro assays.

1. Mixed Lymphocyte Reaction (MLR)

Soluble rCD80.Fc bioactivity was tested in a human Mixed LymphocyteReaction (MLR). Human primary dendritic cells (DC) were generated byculturing monocytes isolated from PBMC (BenTech Bio, USA) in vitro for 7days with 500 U/ml rIL-4 (R&D Systems, USA) and 250 U/ml rGM-CSF (R&DSystems, USA) in Ex-Vivo 15 media (Lonza, Switzerland). 10,000 maturedDC and 100,000 purified allogeneic CD4+ T cells (BenTech Bio, USA) wereco-cultured with variant CD80 Fc fusion proteins and controls in 96 wellround bottom plates in 200 μl final volume of Ex-Vivo 15 media. On day5, IFN-gamma secretion in culture supernatants was analyzed using theHuman IFN-gamma Duoset ELISA kit (R&D Systems, USA). Optical density wasmeasured by VMax ELISA Microplate Reader (Molecular Devices, USA) andquantitated against titrated rIFN-gamma standard included in theIFN-gamma Duo-set kit (R&D Systems, USA).

2. Anti-CD3 Coimmobilization Assay

Costimulatory bioactivity of CD80 fusion variants was determined inanti-CD3 coimmobilization assays. 1 nM or 4 nM mouse anti-human CD3(OKT3, Biolegends, USA) was diluted in PBS with 1 nM to 80 nM rCD80.Fcvariant proteins. This mixture was added to tissue culture treated flatbottom 96-well plates (Corning, USA) overnight to facilitate adherenceof the stimulatory proteins to the wells of the plate. The next day,unbound protein was washed off the plates and 100,000 purified human panT cells (BenTech Bio, US) or human T cell clone BC3 (Astarte Biologics,USA) were added to each well in a final volume of 200 μl of Ex-Vivo 15media (Lonza, Switzerland). Cells were cultured 3 days before harvestingculture supernatants and measuring human IFN-gamma levels with DuosetELISA kit (R&D Systems, USA) as described above.

C. Results

Results for the binding and activity studies for exemplary testedvariants are shown in Tables 7 and 8. In particular, Table 7 indicatesexemplary IgSF domain amino acid substitutions (replacements) in the ECDof CD80 selected in the screen for affinity-maturation against therespective cognate structure CD28. Table 8 indicates exemplary IgSFdomain amino acid substitutions (replacements) in the ECD of CD80selected in the screen for affinity-maturation against the respectivecognate structure PD-L1. For the Tables, the exemplary amino acidsubstitutions are designated by amino acid position number correspondingto the respective reference unmodified ECD sequence. For example, thereference unmodified ECD sequence in Tables 7 and 8 is the unmodifiedCD80 ECD sequence set forth in SEQ ID NO:28. The amino acid position isindicated in the middle, with the corresponding unmodified (e.g.wild-type) amino acid listed before the number and the identifiedvariant amino acid substitution listed after the number. Column 2 setsforth the SEQ ID NO identifier for the variant ECD for each variantECD-Fc fusion molecule.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each variant Fc-fusion molecule tocells engineered to express the cognate counter structure ligand and theratio of the MFI compared to the binding of the corresponding unmodifiedECD-Fc fusion molecule not containing the amino acid substitution(s) tothe same cell-expressed counter structure ligand. The functionalactivity of the variant Fc-fusion molecules to modulate the activity ofT cells also is shown based on the calculated levels of IFN-gamma inculture supernatants (pg/ml) generated either i) with the indicatedvariant ECD-Fc fusion molecule coimmoblized with anti-CD3 or ii) withthe indicated variant ECD-Fc fusion molecule in an MLR assay. Tables 7and Table 8 also depict the ratio of IFN-gamma produced by each variantECD-Fc compared to the corresponding unmodified ECD-Fc in bothfunctional assays.

As shown, the selections resulted in the identification of a number ofCD80 IgSF domain variants that were affinity-modified to exhibitincreased binding for at least one, and in some cases more than one,cognate counter structure ligand. In addition, the results showed thataffinity modification of the variant molecules also exhibited improvedactivities to both increase and decrease immunological activitydepending on the format of the molecule. For example, coimmobilizationof the ligand likely provides a multivalent interaction with the cell tocluster or increase the avidity to favor agonist activity and increase Tcell activation compared to the unmodified (e.g. wildtype) ECD-Fcmolecule not containing the amino acid replacement(s). However, when themolecule is provided as a bivalent Fc molecule in solution, the sameIgSF domain variants exhibited an antagonist activity to decrease T cellactivation compared to the unmodified (e.g. wildtype) ECD-Fv moleculenot containing the amino acid replacement(s).

TABLE 7 Variant CD80 selected against CD28. Molecule sequences, bindingdata, and costimulatory bioactivity data. Coimmobili- zation with anti-MLR CD3 IFN- Binding IFN- gamma SEQ CD28 CTLA- PD-L1 gamma levels ID MFI4 MFI MFI pg/ml pg/ml NO (parental (parental (parental (parental(parental CD80 mutation(s) (ECD) ratio) ratio) ratio) ratio) ratio)L70Q/A91G/N144D 414 125 283 6 93 716 (1.31) (1.36) (0.08) (1.12) (0.83)L70Q/A91G/T130A 56 96 234 7 99 752 (1.01) (1.13) (0.10) (1.19) (0.87)L70Q/A91G/I118A/ 415 123 226 7 86 741 T120S/T130A/K169E (1.29) (1.09)(0.10) (1.03) (0.86) V4M/L70Q/A91G/I118V/ 416 89 263 6 139 991T120S/T130A/K169E (0.94) (1.26) (0.09) (1.67) (1.14)L70Q/A91G/I118V/T120S/ 417 106 263 6 104 741 T130A/K169E (1.12) (1.26)(0.09) (1.25) (0.86) V2OL/L70Q/A91S/Il18V/ T120S/T130A 419 105 200 9 195710 (1.11) (0.96) (0.13) (2.34) (0.82) S44P/L70Q/A91G/T130A 61 88 134 5142 854 (0.92) (0.64) (0.07) (1.71) (0.99) L70Q/A91G/E117G/I118V/ 420120 193 6 98 736 T120S/T130A (1.27) (0.93) (0.08) (1.05) (0.85)A91G/T120S/I118V/T130A 421 84 231 44 276 714 (0.89) (1.11) (0.62) (3.33)(0.82) L7OR/A91G/I118V/T120S/ 422 125 227 6 105 702 T130A/T199S (1.32)(1.09) (0.09) (1.26) (0.81) L70Q/E81A/A91G/I118V/T120S/1127T/ 423 140185 18 98 772 T130A (1.48) (0.89) (0.25) (1.18) (0.89)L70Q/Y87N/A91G/T130A 66 108 181 6 136 769 (1.13) (0.87) (0.08) (1.63)(0.89) T285/L70Q/A91G/ 424 32 65 6 120 834 E95K/I118V/T120S/1126V/(0.34) (0.31) (0.08) (1.44) (0.96) T130A/K169E N635/L70Q/A91G/ 425 124165 6 116 705 S114T/I118V/T120S/T130A (1.30) (0.79) (0.08) (1.39) (0.81)K36E/167T/L70Q/A91G/I118V/T120S/ 426 8 21 5 53 852 T130A/N152T (0.09)(0.10) (0.08) (0.63) (0.98) E52G/L70Q/A91G/D107N/I118V/ 427 113 245 6 94874 T120S/T130A/K169E (1.19) (1.18) (0.08) (1.13) (1.01)K37E/F595/L70Q/A91G/T120S/T130A 428 20 74 6 109 863 (0.21) (0.36) (0.08)(1.31) (1.00) A91G/S103P 72 39 56 9 124 670 (0.41) (0.27) (0.13) (1.49)(0.77) K89E/T130A 73 90 148 75 204 761 (0.95) (0.71) (1.07) (2.45)(0.88) A91G 74 96 200 85 220 877 (1.01) (0.96) (1.21) (2.65) (1.01)D60V/A91G/I118V/T120S/ 429 111 222 12 120 744 T130A/K169E (1.17) (1.07)(0.18) (1.44) (0.86) K54M/L70Q/A91G/Y164H 430 68 131 5 152 685 (0.71)(0.63) (0.08) (1.83) (0.79) M38T/L70Q/E77G/A91G/I118V/T120S/ 431 61 1025 119 796 T130A/N152T (0.64) (0.49) (0.07) (1.43) (0.92)R29H/E52G/L70R/E88G/A91G/T130A 78 100 119 5 200 740 (1.05) (0.57) (0.08)(2.41) (0.85) Y31H/T41G/M43L/L70Q/A91G/ 432 85 85 6 288 782I118V/T120S//I126V/T130A (0.89) (0.41) (0.08) (3.47) (0.90) V68A/T110A80 103 233 48 163 861 (1.08) (1.12) (0.68) (1,96) (0.99)L65H/D90G/T110A/F116L 433 33 121 11 129 758 (0.35) (0.58) (0.15) (1.55)(0.88) R29H/E52G/D9ON/I118V/T120S/T13OA 434 66 141 11 124 800 (0.69)(0.68) (0.15) (1.49) (0.92) A91G/L102S 83 6 6 5 75 698 (0.06) (0.03)(0.08) (0.90) (0.81) 167T/L70Q/A91G/I118V/T120S 436 98 160 5 1751 794(1.03) (0.77) (0.08) (21.1) (0.92) L70Q/A91G/T110A/ 437 8 14 5 77 656I118V/T120S/T130A (0.09) (0.07) (0.07) (0.93) (0.76)M38V/T41D/M431/W50G/D76G/V83A/ 438 5 8 8 82 671K89E/I118V/T120S/I126V/T13OA (0.06) (0.04) (0.11) (0.99) (0.78)V22A/L70Q/S121P 87 5 7 5 105 976 (0.06) (0.04) (0.07) (1.27) (1.13)A12V/S15F/Y31H/M38L/ 439 6 6 5 104 711 T41G/M43L/D9ON/T130A/P137L/(0.06) (0.03) (0.08) (1.25) (0.82) N149D/N152T 167F/L7OR/E88G/A91G/ 4405 6 6 62 1003 I118V/T120S/T130A (0.05) (0.03) (0.08) (0.74) (1.16)E24G/L25P/L70Q/A91G/I118V/T120S/N 441 26 38 8 101 969 152T (0.27) (0.18)(0.11) (1.21) (1.12) A91G/F92L/F108L/I118V/T120S 442 50 128 16 59 665(0.53) (0.61) (0.11) (0.71) (0.77) WT CD80 28 95 208 70 83 866 (1.00)(1.00) (1.00) (1.00) (1.00)

TABLE 8 Variant CD80 selected against PD-Lt. Molecule sequences, bindingdata, and costimulatory bioactivity data. Coimmobilization MLR with IFN-Binding anti-CD3 gamma CD28 CTLA-4 PD-Ll IFN-gamma levels SEQ ID MFI MFIMFI pg/ml pg/ml NO (parental (parental (parental (parental (parentalCD80 mutation(s) (ECD) ratio) ratio) ratio) ratio) ratio)R29D/Y31L/Q33H/ 92 1071 1089 37245 387 5028 K36G/M38I/T41A/ (0.08)(0.02) (2.09) (0.76) (0.26) M43R/M47T/E81V/ L85R/K89N/A91T/F92P/K93V/R94L/ I118T/N149S R29D/Y31L/Q33H/ 93 1065 956 30713 400 7943K36G/M38I/T41A/ (0.08) (0.02) (1.72) (0.79) (0.41) M43R/M47T/E81V/L85R/K89N/A91T/ F92P/K93V/R94L/ N144S/N149S R29D/Y31L/Q33H/ 94 926 95447072 464 17387 K36G/M38I/T41A/ (0.07) (0.02) (2.64) (0.91) (0.91)M42T/M43R/M47T/ E81V/L85R/K89N/ A91T/F92P/K93V/ R94L/L148S/N149SE24G/R29D/Y31L/ 95 1074 1022 1121 406 13146 Q33H/K36G/M38I/ (0.08)(0.02) (0.06) (0.80) (0.69) T41A/M43R/M47T/ F59L/E81V/L85R/K89N/A91T/F92P/ K93V/R94L/H96R/ N149S/C182S R29D/Y31L/Q33H/ 96 1018 97425434 405 24029 K36G/M38I/T41A/ (0.08) (0.02) (1.43) (0.80) (1.25)M43R/M47T/E81V/ L85R/K89N/A91T/ F92P/K93V/R94L/N149S R29V/M43Q/E81R/ 971029 996 1575 342 11695 L851/K89R/D90L/ (0.08) (0.02) (0.09) (0.67)(0.61) A91E/F92N/K93Q/R94G T41I/A91G 98 17890 50624 12562 433 26052(1.35) (1.01) (0.70) (0.85) (1.36) E88D/K89R/D90K/A91G/ 443 41687 4942920140 773 6345 F92Y/K93R/N122S/ (3.15) (0.99) (1.13) (1.52) (0.33) N177SE88D/K89R/D90K/A91G/ 102 51663 72214 26405 1125 9356 F92Y/K93R (3.91)(1.44) (1.48) (2.21) (0.49) K36G/K37Q/M38I/ 445 1298 1271 3126 507 3095L40M/F59L/E81V/L85R/ (0.10) (0.03) (0.18) (1.00) (0.16) K89N/A91T/F92P/K93V/R94L/E99G/ T130A/N149S E88D/K89R/D90K/ 102 31535 50868 29077 9445922 A91G/F92Y/K93R (2.38) (1.02) (1.63) (1.85) (0.31)K36G/K37Q/M38I/L40M 103 1170 1405 959 427 811 (0.09) (0.03) (0.05)(0.84) (0.04) K36G/L40M 443 29766 58889 20143 699 30558 (2.25) (1.18)(1.13) (1.37) (1.59) WTCD80 28 13224 50101 17846 509 19211 (1.00) (1.00)(1.00) (1.00) (1.00)

Example 7 Ligand Binding Competition Assay

As shown in Example 6, several variant CD80 molecules exhibited improvedbinding to one or both of CD28 and PD-L1. To further assess the bindingactivity of CD80 to ligands CD28 and PD-L1, this Example describes aligand competition assay assessing the non-competitive nature ofexemplary variant CD80 polypeptides to bind both CD28 and PD-L1.

An ELISA based binding assay was set up incorporating plate-boundvariant CD80 A91G ECD-Fc to assess the ability of CD80 to simultaneouslybind CD28 and PD-L1. Maxisorp 96 well ELISA plates (Nunc, USA) werecoated overnight with 100 nM human recombinant variant CD80 A91G ECD-Fcfusion protein in PBS. The following day unbound protein was washed out,and the plate was blocked with 1% bovine serum albumin (Millipore,USA)/PBS for 1 hour at room temperature. This blocking reagent waswashed out three times with PBS/0.05% Tween, which included a two minuteincubation on a platform shaker for each wash.

In one arm of the competition assay, CD80 was incubated with CD28, andthen CD28-bound CD80 was then assessed for competitive binding in thepresence of either the other known CD80 ligand counter structures PD-L1or CTLA-4 or negative control ligand PD-L2. Specifically, biotinylatedrecombinant human CD28 Fc fusion protein (rCD28.Fc; R&D Systems) wastitrated into the wells starting at 10 nM, diluting out for eight pointswith 1:2 dilutions in a 25 μl volume. Immediately after adding thebiotinylated rCD28.Fc, unlabeled competitive binders, recombinant humanPD-L1 monomeric his-tagged protein, recombinant human CTLA-4 monomerichis-tagged protein, or a negative control human recombinant PD-L2 Fcfusion protein (R&D Systems) were added to wells at 2000/1000/500 nM,respectively, in a 25 μl volume for a final volume of 50 μl. Theseproteins were incubated together for one hour before repeating the threewash steps as described above.

After washing, 2.5 ng per well of HRP-conjugated streptavidin (JacksonImmunoresearch, USA) were diluted in 1% BSA/PBS and added to wells todetect bound biotinylated rCD28.Fc. After a one hour incubation, wellswere washed again three times as described above. To detect signal, 50μl of TMB substrate (Pierce, USA) were added to the wells following thewash and incubated for 7 minutes, before adding 50 ul 2M sulfuric acidstop solution. Optical density was determined on an Emax Plus microplatereader (Molecular Devices, USA). Optical density values were graphed inPrism (Graphpad, USA).

The results are set forth in FIG. 7A. The results showed decreasedbinding of biotinylated rCD28.Fc to the variant CD80 A91G ECD-Fc fusionprotein with titration of the rCD28.Fc. When rCD28.Fc binding wasperformed in the presence of non-competitive control protein, rPDL2,there was no decrease in CD28 binding for CD80 (solid triangle). Incontrast, a competitive control protein, rCTLA-4, when incubated withthe CD28.Fc, did result in decreased CD28 binding for CD80 as expected(x line). When recombinant PD-L1 was incubated with CD28.Fc, no decreasein CD28 binding to CD80 was observed, which demonstrated that theepitopes of CD28 and PD-L1 for CD80 are non-competitive. Binding of therecombinant PD-L1 protein used in the CD28 competition assay to CD80 wasconfirmed by incubating the biotinylated PD-L1 in the presence ofnon-biotinylated rCD28.Fc (square).

The reverse competition also was set up in which CD80 was incubated withPD-L1, and then PD-L1-bound CD80 was then assessed for competitivebinding in the presence of either the other known CD80 ligand counterstructures CD28 or CTLA-4 or negative control ligand PD-L2.Specifically, the assay was performed by titrating biotinylatedrecombinant human PD-L1-His monomeric protein into wells containing therecombinant variant CD80. Because binding is weaker with this ligand,titrations started at 5000 nM with similar 1:2 dilutions over eightpoints in 25 μL. When the rPD-L1-his was used to detect binding, thecompetitive ligands human rCD28.Fc, human rCTLA-4.Fc, or human rPD-L2.Fccontrol were added at 2.5 nM final concentration in 25 μl for a totalvolume of 50 μl. The subsequent washes, detection, and OD measurementswere the same as described above.

The results are set forth in FIG. 7B. Titrated PD-L1-his binding aloneconfirmed that PD-L1 bound to the variant CD80 A91G ECD-Fc fusionmolecule immobilized on the plate (square). When PD-L1-His binding wasperformed in the presence of non-competitive control protein, rPDL2,there was no decrease in PD-L1 binding for CD80 (triangle). TheCD28-competitive control protein, rCTLA-4, when incubated with thePD-L1-his, did not result in decreased PD-L1 binding for CD80 (x line),even though CTLA-4 is competitive for CD28. This result furtherdemonstrated that lack of competition between CD28 and PD-L1 for CD80binding. Finally, when PD-L1-his was incubated with CD28.Fc, no decreasein PD-L1 binding to CD80 was observed, which demonstrated that theepitopes of CD28 and PD-L1 for CD80 are non-competitive.

Thus, the results showed that CTLA-4, but not PD-L1 or the negativecontrol PD-L2, competed for binding of CD28 to CD80 (FIG. 7A) and thatCD28, CTLA-4, and PD-L2 did not compete for binding of PD-L1 to CD80(FIG. 7B). Thus, these results demonstrated that CD28 and PD-L1 arenon-competitive binders of CD80, and that this non-competitive bindingcan be demonstrated independently of which ligand is being detected inthe ELISA.

Example 8 Additional Affinity Modified IgSF Domains

This example describes the design, creation, and screening of additionalaffinity modified ICOSL, CD86 (B7-2) and NKp30 immunomodulatoryproteins, which are other components of the immune synapse (IS) thathave a demonstrated dual role in both immune activation and inhibition.These examples demonstrate that affinity modification of IgSF domainsyields proteins that can act to both increase and decrease immunologicalactivity. This work also describes the various combinations of thosedomains fused in pairs (i.e., stacked) with a variant affinity modifiedCD80 to form a Type II immunomodulatory protein to achieveimmunomodulatory activity.

Mutant DNA constructs of human ICOSL, CD86 and NKp30 IgSF domains fortranslation and expression as yeast display libraries were generatedsubstantially as described in Example 1. For libraries that targetspecific residues of target protein for complete or partialrandomization with degenerate codons, the coding DNA sequences for theextracellular domains (ECD) of human ICOSL (SEQ ID NO:32) and NKp30 (SEQID NO:54) were ordered from Integrated DNA Technologies (Coralville,Iowa) as a set of overlapping oligonucleotides of up to 80 base pairs(bp) in length. Residues were mutated by targeted mutagenesissubstantially as described in Example 1. Alternatively, random librarieswere constructed to identify variants of the ECD of ICOSL (SEQ IDNO:32), CD86 (SEQ ID NO: 29) and NKp30 (SEQ ID NO:54) substantially asdescribed in Example 1.

The degenerate and random library DNA was introduced into yeastsubstantially as described in Example 2 to generate yeast libraries. Thelibraries were used to select yeast expressing affinity modifiedvariants of ICOSL, CD86 and NKp30 substantially as described in Example3. Cells were processed to reduce non-binders and to enrich for ICOSL,CD86 or NKp30 variants with the ability to bind their exogenousrecombinant counter-structure proteins substantially as described inExample 3. This was then followed by two to three rounds of flowcytometry sorting using exogenous counter-structure protein staining toenrich the fraction of yeast cells that displays improved binders.Magnetic bead enrichment and selections by flow cytometry areessentially as described in Keith D. Miller, 1 Noah B. Pefaur, 2 andCheryl L. Baird1 Current Protocols in Cytometry 4.7.1-4.7.30, July 2008.

With ICOSL, CD86 and NKp30 libraries, target ligand proteins weresourced from R&D Systems (USA) as follows: human rCD28.Fc (i.e.,recombinant CD28-Fc fusion protein), rCTLA4.Fc, rICOS.Fc, and rB7H6.Fc.Two-color flow cytometry was performed substantially as described inExample 3. Yeast outputs from the flow cytometric sorts were assayed forhigher specific binding affinity. Sort output yeast were expanded andre-induced to express the particular IgSF affinity modified domainvariants they encode. This population then can be compared to theparental, wild-type yeast strain, or any other selected outputs, such asthe bead output yeast population, by flow cytometry.

For ICOSL, the second sort outputs (F2) were compared to parental ICOSLyeast for binding of each rICOS.Fc, rCD28.Fc, and rCTLA4.Fc by doublestaining each population with anti-HA (hemagglutinin) tag expression andthe anti-human Fc secondary to detect ligand binding.

In the case of ICOSL yeast variants selected for binding to ICOS, the F2sort outputs gave Mean Fluorescence Intensity (MFI) values of 997, whenstained with 5.6 nM rICOS.Fc, whereas the parental ICOSL strain MFI wasmeasured at 397 when stained with the same concentration of rICOS.Fc.This represents a roughly three-fold improvement of the average bindingin this F2 selected pool of clones, and it is predicted that individualclones from that pool will have much better improved MFI/affinity whenindividually tested.

In the case of ICOSL yeast variants selected for binding to CD28, the F2sort outputs gave MFI values of 640 when stained with 100 nM rCD28.Fc,whereas the parental ICOSL strain MFI was measured at 29 when stainedwith the same concentration of rCD28.Fc (22-fold improvement). In thecase of ICOSL yeast variants selected for binding to CTLA-4, the F2 sortoutputs gave MFI values of 949 when stained with 100 nM rCTLA4.Fc,whereas the parental ICOSL strain MFI was measured at 29 when stainedwith the same concentration of rCTLA4.Fc (32-fold improvement).

In the case of NKp30 yeast variants selected for binding to B7-H6, theF2 sort outputs gave MFI values of 533 when stained with 16.6 nMrB7H6.Fc, whereas the parental NKp30 strain MFI was measured at 90 whenstained with the same concentration of rB7H6.Fc (6-fold improvement).

Among the NKp30 variants that were identified, was a variant thatcontained mutations L30V/A60V/S64P/S86G with reference to positions inthe NKp30 extracellular domain corresponding to positions set forth inSEQ ID NO:54. Among the CD86 variants that were identified, was avariant that contained mutations Q35H/H90L/Q102H with reference topositions in the CD86 extracellular domain corresponding to positionsset forth in SEQ ID NO:29. Among the ICOSL variants that wereidentified, were variants set forth in Table 9 and described furtherbelow.

As with CD80, the MFIs of all F2 outputs described above when measuredwith the anti-HA antibody on FL1 did not increase and sometimes wentdown compared to wild-type strains, indicating that increased bindingwas not a function of increased expression of the selected variants onthe surface of yeast, and validated gating strategies of only selectingmid to low expressors with high ligand binding.

Exemplary selection outputs were reformatted as immunomodulatoryproteins containing an affinity modified (variant) extracellular domain(ECD) of ICOSL fused to an Fc molecule (variant ECD-Fc fusion molecules)substantially as described in Example 4 and the Fc-fusion protein wasexpressed and purified substantially as described in Example 5.

Binding of exemplary ICOSL Fc-fusion variants to cell-expressed counterstructures was then assessed substantially as described in Example 6. Toproduce cells expressing cognate binding partners, full-length mammaliansurface expression constructs for each of human CD28, and ICOS wereproduced substantially as described in Example 6. Binding studies andflow cytometry were carried out substantially as described in Example 6.In addition, the bioactivity of the Fc-fusion variant protein wascharacterized by either mixed lymphocyte reaction (MLR) or anti-CD3coimmobilization assay substantially as described in Example 6.

Results for the binding and activity studies for exemplary testedvariants are shown in Table 9. In particular, Table 9 indicatesexemplary IgSF domain amino acid substitutions (replacements) in the ECDof ICOSL selected in the screen for affinity-maturation against therespective cognate structures ICOS and CD28. The exemplary amino acidsubstitutions are designated by amino acid position number correspondingto the respective reference unmodified ECD sequence as follows. Forexample, the reference unmodified ECD sequence in Table 9 is theunmodified ICOSL ECD sequence set forth in SEQ ID NO:32. The amino acidposition is indicated in the middle, with the corresponding unmodified(e.g. wild-type) amino acid listed before the number and the identifiedvariant amino acid substitution listed after the number. Column 2 setsforth the SEQ ID NO identifier for the variant ECD for each variantECD-Fc fusion molecule.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each variant Fc-fusion molecule tocells engineered to express the cognate counter structure ligand and theratio of the MFI compared to the binding of the corresponding unmodifiedECD-Fc fusion molecule not containing the amino acid substitution(s) tothe same cell-expressed counter structure ligand. The functionalactivity of the variant Fc-fusion molecules to modulate the activity ofT cells also is shown based on the calculated levels of IFN-gamma inculture supernatants (pg/ml) generated either i) with the indicatedvariant ECD-Fc fusion molecule coimmoblized with anti-CD3 or ii) withthe indicated variant ECD-Fc fusion molecule in an MLR assay. The Tablesalso depict the ratio of IFN-gamma produced by each variant ECD-Fccompared to the corresponding unmodified ECD-Fc in both functionalassays.

As shown, the selections resulted in the identification of a number ofICOSL IgSF domain variants that were affinity-modified to exhibitincreased binding for at least one, and in some cases more than one,cognate counter structure ligand. In addition, the results showed thataffinity modification of the variant molecules also exhibited improvedactivities to both increase and decrease immunological activitydepending on the format of the molecule. For example, coimmobilizationof the ligand likely provides a multivalent interaction with the cell tocluster or increase the avidity to favor agonist activity and increase Tcell activation compared to the unmodified (e.g. wildtype) ECD-Fcmolecule not containing the amino acid replacement(s). However, when themolecule is provided as a bivalent Fc molecule in solution, the sameIgSF domain variants exhibited an antagonist activity to decrease T cellactivation compared to the unmodified (e.g. wildtype) ECD-Fv moleculenot containing the amino acid replacement(s).

TABLE 9 ICOSL variants selected against CD28 or ICOS. Moleculesequences, binding data, and costimulatory bioactivity data. Coimmobi-lization with anti- MLR CD3 IFN- Binding IFN- gamma SEQ ICOS CD28 gammalevels ID OD MFI pg/ml pg/ml ICOSL NO (parental (parental (parental(parental mutation(s) (ECD) ratio) ratio) ratio) ratio) N52S 109 1.33(1.55) 162 (9.00) 1334 (1.93) 300 (0.44) N52H 110 1.30 (1.51) 368(20.44) 1268 (1.83) 39 (0.06) N52D 111 1.59 (1.85) 130 (7.22) 1943(2.80) 190 (0.28) N52Y/N57Y/ 112 1.02 (1.19) 398 (22.11) 510* (1.47*) 18(0.03) F138L/L203P N52H/N57Y/ 113 1.57 (1.83) 447 (24.83) 2199 (3.18) 25(0.04) Q100P N52S/Y146C/ 114 1.26 (1.47) 39 (2.17) 1647 (2.38) 152(0.22) Y152C N52H/C198R 115 1.16 (1.35) 363 (20.17) 744* (2.15*) ND (ND)N52H/C140del/ 478 ND (ND) 154 (8.56) 522* (1.51*) ND (ND) T225AN52H/C198R/ 117 1.41 (1.64) 344 (19.11) 778* (2.25*) 0 (0) T225AN52H/K92R 118 1.48 (1.72) 347 (19.28) 288* (0.83*) 89 (0.13) N52H/S99G119 0.09 (0.10) 29 (1.61) 184* (0.53*) 421 (0.61) N52Y 120 0.08 (0.09)18 (1.00) 184* (0.53*) 568 (0.83) N57Y 121 1.40 (1.63) 101 (5.61) 580*(1.68*) 176 (0.26) N57Y/Q100P 122 0.62 (0.72) 285 (15.83) 301* (0.87*)177 (0.26) N52S/S130G/ 123 0.16 (0.19) 24 (1.33) 266* (0.77*) 1617(2.35) Y152C N52S/Y152C 124 0.18 (0.21) 29 (1.61) 238* (0.69*) 363(0.53) N52S/C198R 125 1.80 (2.09) 82 (4.56) 1427 (2.06) 201 (0.29)N52Y/N57Y/ 126 0.08 (0.09) 56 (3.11) 377* (1.09*) 439 (0.64) Y152CN52Y/N57Y/ 127 ND (ND) 449 (24.94) 1192 (1.72) ND (ND) H129P/C198RN52H/L161P/ 128 0.18 (0.21) 343 (19.05) 643* (1.86*) 447 (0.65) C198RN52S/T113E 129 1.51 (1.76) 54 (3.00) 451* (1.30*) 345 (0.50) S54A 1301.62 (1.88) 48 (2.67) 386* (1.12*) 771 (1.12) N52D/S54P 131 1.50 (1.74)38 (2.11) 476* (1.38*) 227 (0.33) N52K/L208P 132 1.91 (2.22) 291 (16.17)1509 (2.18) 137 (0.20) N52S/Y152H 133 0.85 (0.99) 68 (3.78) 2158 (3.12)221 (0.32) N52D/V151A 134 0.90 (1.05) 19 (1.06) 341* (0.99*) 450 (0.66)N52H/I143T 135 1.83 (2.13) 350 (19.44) 2216 (3.20) 112 (0.16) N52S/L80P136 0.09 (0.10) 22 (1.22) 192* (0.55*) 340 (0.49) F120S/Y152H/ 137 0.63(0.73) 16 (0.89) 351* (1.01*) 712 (1.04) N201S N52S/R75Q/ 138 1.71(1.99) 12 (0.67) 1996 (2.88) 136 (0.20) L203P N52S/D158G 139 1.33 (1.55)39 (2.17) 325* (0.94*) 277 (0.40) N52D/Q133H 140 1.53 (1.78) 104 (5.78)365* (1.05*) 178 (0.26) WT ICOSL 32 0.86 (1.00) 18 (1.00) 692/346*(1.00) 687 (1.00) *Parental ratio calculated using 346 ng/ml IFN-gammafor WT ICOSL

Example 9 Generation and Assessment of Stacked Molecules ContainingDifferent Affinity-Modified Domains

This Example describes further immunomodulatory proteins that weregenerated as stack constructs containing at least two different affinitymodified domains from identified variant CD80 polypeptides and one moreadditional variant CD80, CD86, ICOSL, and NKp30 molecules linkedtogether and fused to an Fc.

Selected variant molecules described above that were affinity-modifiedfor one or more counter structure ligand were used to generate “stack”molecule (i.e., Type II immunomodulatory protein) containing two or moreaffinity-modified IgSF domains. Stack constructs were obtained asgeneblocks (Integrated DNA Technologies, Coralville, Iowa) that encodethe stack in a format that enables its fusion to Fc by standard Gibsonassembly using a Gibson assembly kit (New England Biolabs).

The encoding nucleic acid molecule of all stacks was generated to encodea protein designed as follows: Signal peptide, followed by the firstvariant IgV of interest, followed by a 15 amino acid linker which iscomposed of three GGGGS(G4S) motifs (SEQ ID NO:228), followed by thesecond IgV of interest, followed by two GGGGS linkers (SEQ ID NO: 229)followed by three alanines (AAA), followed by a human IgG1 Fc asdescribed above. To maximize the chance for correct folding of the IgVdomains in each stack, the first IgV was preceded by all residues thatnormally occur in the wild-type protein between this IgV and the signalpeptide (leading sequence). Similarly, the first IgV was followed by allresidues that normally connect it in the wild-type protein to either thenext Ig domain (typically an IgC domain) or if such a second IgV domainis absent, the residues that connect it to the transmembrane domain(trailing sequence). The same design principle was applied to the secondIgV domain except that when both IgV domains were derived from sameparental protein (e.g. a CD80 IgV stacked with another CD80 IgV), thelinker between both was not duplicated.

Table 10 sets forth the design for exemplary stacked constructs. Theexemplary stack molecules shown in Table 10 contain the IgV domains asindicated and additionally leading or trailing sequences as describedabove. In the Table, the following components are present in order:signal peptide (SP; SEQ ID NO:225), IgV domain 1 (IgV1), trailingsequence 1 (TS1), linker 1 (LR1; SEQ ID NO:228), IgV domain 2 (IgV2),trailing sequence 2 (TS2), linker 2 (LR2; SEQ ID NO:230) and Fc domain(SEQ ID NO:226 containing C5S/R77C/N82GN87C amino acid substitutions).In some cases, a leading sequence 1 (LS1) is present between the signalpeptide and IgV1 and in some cases a leading sequence 2 (LS2) is presentbetween the linker and IgV2.

TABLE 10 Amino acid sequence (SEQ ID NO) of components of exemplarystacked constructs First domain Second domain SP LS1 IgV1 TS1 LR1 L52IgV2 T52 LR2 Fc Domain 1: NKp30 + − 214 235 + − 152 371 + + WT Domain 2:CD80 WT Domain 1: NKp30 + − 214 235 + 236 220 237 + + WT Domain 2: CD86WT Domain 1: NKp30 + − 215 235 + − 192 371 + + L30V/A60V/S64P/ S86GDomain 2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D9ON/A91G/P109S Domain 1:NKp30 + − 215 235 + − 175 371 + + L30V/A60V/S64P/ S86G Domain 2: CD80I67T/L70Q/A91G/ T120S Domain 1: CD80 + − 152 371 + − 214 235 + + WTDomain 2: Nkp30 WT Domain 1: CD80 + − 192 371 + − 215 235 + +R29H/Y31H/T41G/ Y87N/E88G/K89E/ D9ON/A91G/P109S Domain 2. NKp30L30V/A60V/S64P/ S86G Domain 1: CD80 + − 175 371 + − 215 235 + +I67T/L70Q/A91G/ T120S Domain 2. NKp30 L30V/A60V/S64P/ S86G Domain 1:CD80 + − 152 371 + − 196 233 + + WT Domain 2: ICOSL WT Domain 1: CD80 +− 152 371 + 236 220 237 + + WT Domain 2: CD86 WT Domain 1: CD80 + − 152371 + − 152 371 + + WT Domain 2: CD80 WT Domain 1: CD80 + − 189 371 + −192 371 + + E88D/K89R/D90K/ A91G/F92Y/K93R Domain 2: CD80R29H/Y31H/T41G/ Y87N/E88G/K89E/ D9ON/A91G/P109S Domain 1: CD80 + − 193371 + − 192 371 + + A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2:CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D9ON/A91G/P109S Domain 1: CD80 + −193 371 + − 175 371 + + A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2:CD80 I67T/L70Q/A91G/ T120S Domain 1: CD80 + − 189 371 + 236 221 237 + +E88D/K89R/D90K/ A91G/F92Y/K93R Domain 2: CD86 Q35H/H9OL/Q102H Domain 1:CD80 + − 193 371 + 236 221 237 + + A12T/H18L/M43V/ F59L/E77K/P109S/I118T Domain 2: CD86 Q35H/H9OL/Q102H Domain 1: CD80 + − 189 371 + − 213233 + + E88D/K89R/D90K/ A91G/F92Y/K93R Domain 2: ICOSL N52S/N57Y/H94D/L96F/L98F/Q100 R/G103E/ F120S Domain 1: CD80 + − 193 371 + − 213 233 + +A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2: ICOSL N52S/N57Y/H94D/L96F/L98F/Q100R/ G103E/ F120S Domain 1: CD80 + − 193 371 + − 199 233 + +A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2: ICOSL N52D Domain 1:CD80 + − 189 371 + − 201 233 + + E88D/K89R/D90K/ A91G/F92Y/K93R Domain2: ICOSL N52H/N57Y/Q100P Domain 1: CD80 + − 193 371 + − 201 233 + +A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2: ICOSL N52H/N57Y/Q100PDomain 1: ICOSL + − 196 233 + − 152 371 + + WT Domain 2. CD80 WT Domain1: CD86 + 236 220 237 + − 152 371 + + WT Domain 2. CD80 WT Domain 1:CD80 + − 192 371 + − 189 371 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/D9ON/A91G/P109S Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1:CD80 + − 192 371 + − 193 371 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/D90N/A91G/P109S Domain 2: CD80 A12T/H18L/M43V/ F59L/E77K/P109S/ I118TDomain 1: CD80 + − 175 371 + − 189 371 + + I67T/L70Q/A91G/ T120S Domain2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1: CD80 + − 175 371 + −193 371 + + I67T/L70Q/A91G/ T120S Domain 2: CD80 A12T/H18L/M43V/F59L/E77K/P109S/ I118T Domain 1: CD86 + 236 221 237 + − 189 371 + +Q35H/H90L/Q102H Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1:CD86 + 236 221 237 + − 193 371 + + Q35H/H9OL/Q102H Domain 2: CD80A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 1: ICOSL + − 213 233 + −189 371 + + N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/ F120S Domain 2: CD80E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1: ICOSL + − 213 233 + − 193371 + + N52S/N57Y/H94D/ L96F/L98F/Q100 R/G103E/ F120S Domain 2: CD80A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 1: ICOSL + − 199 233 + −189 371 + + N52D Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1:ICOSL + − 199 233 + − 193 371 + + N52D Domain 2: CD80 A12T/H18L/M43V/F59L/E77K/P109S/ I118T Domain 1: ICOSL + − 201 233 + − 189 371 + +N52H/N57Y/Q100P Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1:ICOSL + − 201 233 + − 193 371 + + N52H/N57Y/Q100P Domain 2: CD80A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 1: CD80 + − 195 371 + −189 371 + + V68M/L70P/L72P/ K86E Domain 2: CD80 E88D/K89R/D90K/A91G/F92Y/K93R Domain 1: CD80 + − 194 371 + − 189 371 + +R29V/Y31F/K36G/ M38L/M43Q/E81R/ V83I/L851/K89R/ D90L/A91E/F92N/K93Q/R94G Domain 2: CD80 E88D/K89R/D90K/ A91G/F92Y/K93R Domain 1: CD80 +− 195 371 + − 193 371 + + V68M/L70P/L72P/ K86E Domain 2: CD80A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 1: CD80 + − 194 371 + −193 371 + + R29V/Y31F/K36G/ M38L/M43Q/E81R/ V83I/L851/K89R/D9OL/A91E/F92N/ K93Q/R94G Domain 2: CD80 A12T/H18L/M43V/F59L/E77K/P109S/ I118T Domain 1: CD80 + − 189 371 + − 195 371 + +E88D/K89R/D90K/ A91G/F92Y/K93R Domain 2: CD80 V68M/L70P/L72P/ K86EDomain 1: CD80 + − 189 371 + − 194 371 + + E88D/K89R/D90K/A91G/F92Y/K93R Domain 2: CD80 R29V/Y31F/K36G/ M38L/M43Q/E81R/V83I/L851/K89R/ D90L/A91E/F92N/ K93Q/R94G Domain 1: CD80 + − 193 371 + −195 371 + + A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2: CD80V68M/L70P/L72P/ K86E Domain 1: CD80 + − 193 371 + − 194 371 + +A12T/H18L/M43V/ F59L/E77K/P109S/ I118T Domain 2: CD80 R29V/Y31F/K36G/M38L/M43Q/E81R/ V83I/ L85I/K89R/D90L/ A91E/F92N/K93Q/ R94G Domain 1:CD80 + − 192 371 + − 213 233 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/D9ON/A91G/P109S Domain 2: ICOSL N52S/N57Y/H94D/ L96F/L98F/Q100 R/G103E/F120S Domain 1: CD80 + − 175 371 + − 213 233 + + I67T/L70Q/A91G/ T120SDomain 2: ICOSL N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/ F120S Domain 1:CD80 + − 192 371 + − 199 233 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/D9ON/A91G/P109S Domain 2: ICOSL N52D Domain 1: CD80 + − 175 371 + − 199233 + + I67T/L70Q/A91G/ T120S Domain 2: ICOSL N52D Domain 1: CD80 + −192 371 + − 201 233 + + R29H/Y31H/T41G/ Y87N/E88G/K89E/ D9ON/A91G/P109SDomain 2: ICOSL N52H/N57Y/Q100P Domain 1: CD80 + − 175 371 + − 201233 + + I67T/L70Q/A91G/ T120S Domain 2: ICOSL N52H/N57Y/Q100P Domain 1:ICOSL + − 213 233 + − 192 371 + + N52S/N57Y/H94D/ L96F/L98F/Q100R/G103E/ F120S Domain 2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/D9ON/A91G/P109S Domain 1: ICOSL + − 213 233 + − 175 371 + +N52S/N57Y/H94D/ L96F/L98F/Q100R/ G103E/ F120S Domain 2: CD80I67T/L70Q/A91G/ T120S Domain 1: ICOSL + − 199 233 + − 192 371 + + N52DDomain 2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D9ON/A91G/P109S Domain 1:ICOSL + − 199 233 + − 175 371 + + N52D Domain 2: CD80 I67T/L70Q/A91G/T120S Domain 1: ICOSL + − 201 233 + − 192 371 + + N52H/N57Y/Q100P Domain2: CD80 R29H/Y31H/T41G/ Y87N/E88G/K89E/ D90N/A91G/P109S

High throughput expression and purification of the variantIgV-stacked-Fc fusion molecules containing various combinations ofvariant IgV domains from CD80, CD86, ICOSL or Nkp30 containing at leastone affinity-modified IgV domain were generated substantially asdescribed in Example 5. Binding of the variant IgV-stacked-Fc fusionmolecules to respective counter structures and functional activity byanti-CD3 coimmobilization assay also were assessed substantially asdescribed in Example 6. For example, costimulatory bioactivy of thestacked IgSF Fc fusion proteins was determined in a similar immobilizedanti-CD3 assay as above. In this case, 4 nM of anti-CD3 (OKT3,Biolegend, USA) was coimmobilized with 4 nM to 120 nM of human rB7-H6.Fc(R&D Systems, USA) or human rPD-L1.Fc (R&D Systems, USA) overnight ontissue-culture treated 96-well plates (Corning, USA). The following dayunbound protein was washed off with PBS and 100,000 purified pan T cellswere added to each well in 100 ul Ex-Vivo 15 media (Lonza, Switzerland).The stacked IgSF domains were subsequently added at concentrationsranging from 8 nM to 40 nM in a volume of 100 μl for 200 μl volumetotal. Cells were cultured 3 days before harvesting culture supernatantsand measuring human IFN-gamma levels with Duoset ELISA kit (R&D Systems,USA) as mentioned above.

The results are set forth in Tables 11-14. Table 11 sets forth bindingand functional activity results for variant IgV-stacked-Fc fusionmolecules containing an Nkp30 IgV domain and a CD80 or CD86 IgV domain.Table 12 sets forth binding and functional activity results for variantIgV-stacked-Fc fusion molecules containing a variant CD80 IgV domain anda CD80, CD86 or ICOSL IgV domain. Table 13 sets forth binding andfunctional activity results for variant IgV-stacked-Fc fusion moleculescontaining two variant CD80 IgV domains. Table 14 sets forth results forvariant IgV-stacked Fc fusion molecules containing a variant CD80 IgVdomain and a variant ICOSL IgV domain.

For each of Tables 11-14, Column 1 indicates the structural organizationand orientation of the stacked, affinity modified or wild-type (WT)domains beginning with the amino terminal (N terminal) domain, followedby the middle WT or affinity modified domain located before the Cterminal human IgG1 Fc domains. Column 2 sets forth the SEQ ID NOidentifier for the sequence of each IgV domain contained in a respective“stack” molecule. Column 3 shows the binding partners which theindicated affinity modified stacked domains from column 1 were selectedagainst.

Also shown is the binding activity as measured by the Mean FluorescenceIntensity (MFI) value for binding of each stack molecule to cellsengineered to express various counter structure ligands and the ratio ofthe MFI compared to the binding of the corresponding stack moleculecontaining unmodified IgV domains not containing the amino acidsubstitution(s) to the same cell-expressed counter structure ligand. Thefunctional activity of the variant stack molecules to modulate theactivity of T cells also is shown based on the calculated levels ofIFN-gamma in culture supernatants (pg/ml) generated with the indicatedvariant stack molecule in solution and the appropriate ligandcoimmoblized with anti-CD3 as described in Example 6. The Table alsodepicts the ratio of IFN-gamma produced by each variant stack moleculecompared to the corresponding unmodified stack molecule in thecoimmobilization assay.

As shown, the results showed that it was possible to generate stackmolecules containing at least one variant IgSF domain that exhibitedaffinity-modified activity of increased binding for at least one cognatecounter structure ligand compared to a corresponding stack moleculecontaining the respective unmodified (e.g. wild-type) IgV domain. Insome cases, the stack molecule, either from one or a combination of bothvariant IgSF domains in the molecule, exhibited increased binding formore than one cognate counter structure ligand. The results also showedthat the order of the IgV domains in the stacked molecules could, insome cases, alter the degree of improved binding activity. In somecases, functional T cell activity also was altered when assessed in thetargeted coimmobilization assay.

TABLE 11 Stacked variant IgV Fc fusion proteins containing an NKp30 IgVdomain and a CD80 or CD86 IgV domain Anti- CD3 coimmobi- lization assayBinding pg/ml Activity IFN- B7H6 CD28 gamma MFI MFI (WT SEQ Counter (WT(WT parental Domain Structure ID structure parental parental IFN- Nterminal to C terminal: NO selected MFI MFI gamma domain 1/domain 2/Fc(IgV) against ratio) ratio) ratio) Domain 1: NKp30 WT 214 — 88823 (1.00)7022 (1.00) 68 (1.00) Domain 2: CD80 WT 152 Domain 1: NKp30 WT 214 —14052 (1.00) 1690 (1.00) 92 (1.00) Domain 2: CD86 WT 220 Domain 1: NKp30(L30V 215 B7-H6 53279 (0.60) 9027 (1.29) 94 (1.38) A60V S64P S86G)Domain 2: CD80 192 CD28 R29H/Y31H/T41G/Y87N/ E88G/K89E/D90N/A91G/ P109SDomain 1: NKp30 (L30V 215 B7-H6 41370 (0.47) 11240 (1.60) 60 (0.88) A60VS64P S86G) Domain 2: CD80 175 CD28 I67T/L70Q/A91G/T120S Domain 1: NKp30(L30V 215 B7-H6 68480 (4.87) 9115 (5.39) 110 (1.19) A60V S64P S86G)/Domain 2: CD86 221 CD28 Q35H/H90L/Q102H Domain 1: CD80 WT 152 — 110461(1.00) 13654 (1.00) 288 (1.00) Domain 2: NKp30 WT 214 Domain 1: CD86 WT220 CD28 128899 (1.00) 26467 (1.00) 213 (1.00) Domain 2: NKp30 WT 214B7-H6 Domain 1: CD80 192 CD28 55727 (0.50) 4342 (0.32) 100 (0.35)R29H/Y31H/T41G/Y87N/ E88G/K89E/D90N/ A91G/P109S Domain 2: NKp30 (L30V215 B7-H6 A60V S64P S86G) Domain 1: CD80 175 CD28 40412 (0.37) 7094(0.52) 84 (0.29) I67T/L70Q/A91G/T120S Domain 2: NKp30 (L30V 215 B7-H6A60V S64P S86G) Domain 1: CD86 221 CD28 220836 (0.53) 11590 (1.71) 113(0.44) Q35H/H90L/Q102H Domain 2: NKp30 (L30V 215 B7-H6 A60V S64P S86G)

TABLE 12 Stacked variant IgV Fc fusion proteins containing a CD80 IgVdomain and a CD80, CD86, or ICOSL IgV domain Anti-CD3 coimmobili- zationBinding Activity assay PD-L1 ICOS pg/ml MFI MFI IFN- Counter CD28 MFI(WT (WT gamma Domain Structure SEQ ID structure (WT parental parental(WT parental N terminal to C terminal: NO selected parental MFI MFIIFN-gamma domain 1/domain 2/Fc (IgV) against MFI ratio) ratio) ratio)ratio) Domain 1: CD80 WT 152 1230 2657 11122 69 Domain 2: ICOSL WT 196(1.00) (1.00) (1.00) (1.00) Domain 1: CD80 WT 152 60278 2085 59 Domain2: CD86 WT 220 (1.00) (1.00) (1.00) Domain 1: CD80 WT 152 1634 6297 98Domain 2: CD80 WT 152 (1.00) (1.00) (1.00) Domain 1: CD80 189 PD-L1 43084234 214 E88D/K89R/D90K/A91G/F9 192 CD28 (2.64) (0.67) (2.18) 2Y/K93RDomain 2: CD80 R29H/Y31H/T41G/Y87N/E8 8G/K89E/D9ON/A91G/P109S Domain 1:CD80 193 PD-L1 7613 2030 137 A12T/H18L/M43V/F59L/E7 192 CD28 (4.66)(0.32) (1.40) 7K/P109S/I118T Domain 2: CD80 R29H/Y31H/T41G/Y87N/E88G/K89E/D9ON/A91G/P109S Domain 1: CD80 193 PD-L1 3851 3657 81A12T/H18L/M43V/F59L/E7 175 CD28 (2.36) (0.58) (0.83) 7K/P109S/I118TDomain 2: CD80 I67T/L70Q/A91G/T120S Domain 1: CD80 189 PD-L1 4117 291496 E88D/K89R/D9OK/A91G/F9 221 CD28 (0.07) (1.40) (1.63) 2Y/K93R Domain2: CD86 Q35H/H9OL/Q102H Domain 1: CD80 193 PD-L1 2868 3611 94A12T/H18L/M43V/F59L/E7 221 CD28 (0.05) (1.73) (1.60) 7K/P109S/I118TDomain 2: CD86 Q35H/H9OL/Q102H Domain 1: CD80 189 PD-L1 3383 4515 515890 E88D/K89R/D9OK/A91G/F9 213 ICOS/CD28 (2.75) (1.70) (0.46) (1.30)2Y/K93R Domain 2: ICOSL N52S/N57Y/H94D/L96F/L9 8F/Q100R/G103E/ F120SDomain 1: CD80 193 PD-L1 2230 2148 3860 112 A12T/H18L/M43V/F59L/E7 213ICOS/CD28 (1.81) (0.81) (0.35) (1.62) 7K/P109S/I118T Domain 2: ICOSLN52S/N57Y/H94D/L96F/L9 8F/Q100R/G103E/ F120S Domain 1: CD80 193 PD-L15665 6446 15730 126 A12T/H18L/M43V/F59L/E7 199 ICOS/CD28 (4.61) (2.43)(1.41) (1.83) 7K/P109S/I118T Domain 2: ICOSL N52D Domain 1: CD80 189PD-Ll 6260 4543 11995 269 E88D/K89R/D9OK/A91G/F9 201 ICOS/CD28 (5.09)(1.71) (1.08) (3.90) 2Y/K93R Domain 2: ICOSL N52H/N57Y/Q10013 Domain 1:CD80 193 PD-L1 3359 3874 8541 97 A12T/H18L/M43V/F59L/E7 201 ICOS/CD28(2.73) (1.46) (0.77) (1.41) 7K/P109S/I118T Domain 2: ICOSLN52H/N57Y/Q100P Domain 1: ICOSL WT 196 3000 2966 14366 101 Domain 2:CD80 WT 152 (1.00) (1.00) (1.00) (1.00) Domain 1: CD86 WT 220 4946 1517125 Domain 2: CD80 WT 152 (1.00) (1.00) (1.00) Domain 1: CD80 192 CD282832 3672 114 R29H/Y31H/T41G/Y87N/E8 189 PD-L1 (1.73) (0.58) (1.16)8G/K89E/D9ON/A91G/P109S Domain 2: CD80 E88D/K89R/D90K/A91G/F9 2Y/K93RDomain 1: CD80 192 CD28 4542 2878 142 R29H/Y31H/T41G/Y87N/E8 193 PD-L1(2.78) (0.45) (1.45) 8G/K89E/D90N/A91G/P109S Domain 2: CD80A12T/H18L/M43V/F59L/E7 7K/P109S/1118T Domain 1: CD80 175 CD28 938 995102 167T/L70Q/A91G/T120S 189 PD-L1 (0.57) (0.16) (1.04) Domain 2: CD80E88D/K89R/D9OK/A91G/F9 2Y/K93R Domain 1: CD80 175 CD28 4153 2827 108167T/L70Q/A91G/T120S 193 PD-L1 (2.54) (0.45) (1.10) Domain 2: CD80A12T/H18L/M43V/F59L/E7 7K/P109S/1118T Domain 1: CD86 221 CD28 14608 2535257 Q35H/H9OL/Q102H 189 PD-L1 (2.95) (1.67) (2.06) Domain 2: CD80E88D/K89R/D9OK/A91G/F9 2Y/K93R Domain 1: CD86 221 CD28 2088 2110 101Q35H/H9OL/Q102H 193 PD-L1 (0.42) (1.39) (0.81) Domain 2: CD80A12T/H18L/M43V/F59L/E7 7K/P109S/1118T Domain 1: ICOSL 213 ICOS/CD28 36344893 6403 123 N52S/N57Y/H94D/L96F/L9 189 (1.21) (1.65) (0.45) (1.22)8F/Q100R/G103E/ F120S PD-L1 Domain 2: CD80 E88D/K89R/D90K/A91G/F92Y/K93R Domain 1: ICOSL 213 ICOS/CD28 1095 5929 7923 127N52S/N57Y/H94D/L96F/L9 193 (0.37) (2.0) (0.55) (1.26) 8F/Q100R/G103E/F120S PD-L1 Domain 2: CD80 A12T/H18L/M43V/F59L/E7 7K/P109S/I118T Domain1: ICOSL 199 ICOSL/CD28 2023 5093 16987 125 N52D 189 PD-L1 (0.67) (1.72)(1.18) (1.24) Domain 2: CD80 E88D/K89R/D9OK/A91G/F9 2Y/K93R Domain 1:ICOSL 199 ICOS/CD28 3441 3414 20889 165 N52D 193 (1.15) (1.15) (1.45)(1.63) PD-L1 Domain 2: CD80 A12T/H18L/M43V/F59L/E7 7K/P109S/I118T Domain1: ICOSL 201 ICOS/CD28 7835 6634 20779 95 N52H/N57Y/Q10013 189 (2.61)(2.24) (1.45) (0.94) PD-L1 Domain 2: CD80 E88D/K89R/D9OK/A91G/F9 2Y/K93RDomain 1: ICOSL 201 ICOS/CD28 8472 3789 13974 106 N52H/N57Y/Q10013 193(2.82) (1.28) (0.97) (1.05) PD-L1 Domain 2: CD80 A12T/H18L/M43V/F59L/E77K/P109S/I118T

TABLE 13 Stacked variant IgV Fc fusion proteins containing two CD80 IgVdomains Binding Activity PD-L1 CTLA-4 Functional MFI MFI Activity SEQCounter (WT (WT MLR DomainStructure ID structure parental parental IFN-N terminal to C terminal: NO selected MFI MFI gamma domain 1/domain 2/Fc(IgV) against ratio) ratio) pg/ml Domain 1: CD80 WT 152 6297 (1.00) 4698(1.00) 35166 (1.00) Domain 2: CD80 WT 152 Domain 1: CD80 195 CTLA-4 2464(0.39) 4955 (1.05) 5705 (0.16) V68M/L70P/L72P/K86E Domain 2: CD80 189PD-L1 E88D/K89R/D90K/A91G/ F92Y/K93R Domain 1: CD80 194 CTLA-4 1928(0.31) 1992 (0.42) 1560 (0.04) R29V/Y31F/K36G/M38L/ M43Q/E81R/V83I/L85I/K89R/D90L/A91E/F92N/ K93Q/R94G Domain 2: CD80 189 PD-L1 E88D/K89R/D90K/A91G/F92Y/K93R Domain 1: CD80 195 CTLA-4 1215 (0.19) 1382 (0.29) 2171(0.06) V68M/L70P/L72P/K86E Domain 2: CD80 193 PD-L1 A12T/H18L/M43V/F59L/E77K/P109S/I118T Domain 1: CD80 194 CTLA-4 1592 (0.25) 1962 (0.42) 1512(0.04) R29V/Y31F/K36G/M38L/ M43Q/E81R/V83I/L85I/ K89R/D90L/A91E/F92N/K93Q/R94G Domain 2: CD80 193 PD-L1 A12T/H18L/M43V/F59L/ E77K/P109S/I118TDomain 1: CD80 189 PD-L1 1747 (0.28) 2057 (0.44) 9739 (0.28)E88D/K89R/D90K/ A91G/F92Y/K93R Domain 2: CD80 195 CTLA-4V68M/L70P/L72P/K86E Domain 1: CD80 189 PD-L1 1752 (0.28 ) 1772 (0.38)5412 (0.15) E88D/K89R/D90K/ A91G/F92Y/K93R Domain 2: CD80 194 CTLA-4R29V/Y31F/K36G/M38L/ M43Q/E81R/V83I/L85I/ K89R/D90L/A91E/ F92N/K93Q/R94GDomain 1: CD80 193 PD-L1 1636 (0.26) 1887 (0.40) 7608 (0.22)A12T/H18L/M43V/F59L/ E77K/P109S/I118T Domain 2: CD80 195 CTLA-4V68M/L70P/L72P/K86E Domain 1: CD80 193 PD-L1 2037 (0.32) 4822 (1.03)11158 (0.32) A12T/H18L/M43V/F59L/ E77K/P109S/I118T Domain 2: CD80 194CTLA-4 R29V/Y31F/K36G/M38L/ M43Q/E81R/V83I/ L85I/K89R/D90L/A91E/F92N/K93Q/R94G

TABLE 14 Stacked variant IgV Fc fusion proteins containing a CD80 IgVdomain and an ICOSL IgV domain Binding Activity PD-L1 CTLA-4 FunctionalMFI MFI Activity SEQ Counter (WT (WT MLR DomainStructure ID structureparental parental IFN- N terminal to C terminal: NO selected MFI MFIgamma domain 1/domain 2/Fc (IgV) against ratio) ratio) pg/ml Domain 1:CD80 WT 152 1230 (1.00) 11122 (1.00) 1756 (1.00) Domain 2: ICOSL WT 196Domain 1: CD80 192 CD28 2280 (1.85) 3181 (0.29) 2281 (1.30)R29H/Y31H/T41G/Y87N/ E88G/K89E/D90N/A91G/ P109S Domain 2: ICOSL 213ICOS/ N52S/N57Y/H94D/ CD28 L96F/L98F/Q100R/ G103E/F120S Domain 1: CD80175 CD28 2309 (1.88) 26982 (2.43) 1561 (0.89) I67T/L70Q/A91G/T120SDomain 2: ICOSL 213 ICOS/ N52S/N57Y/H94D/ CD28 L96F/L98F/Q100R/G103E/F120S Domain 1: CD80 192 CD28 4285 (3.48) 22744 (2.04) 1612 (0.92)R29H/Y31H/T41G/Y87N/ E88G/K89E/D90N/ A91G/P109S Domain 2: ICOSL 199ICOS/ N52D CD28 Domain 1: CD80 175 CD28 3024 (2.46) 16916 (1.52) 3857(2.20) I67T/L70Q/A91G/T120S Domain 2: ICOSL 199 ICOS/ N52D CD28 Domain1: CD80 192 CD28 6503 (5.29) 7240 (0.65) 6886 (3.92) R29H/Y31H/T41G/Y87N/E88G/K89E/ D90N/A91G/P109S Domain 2: ICOSL 201 ICOS/N52H/N57Y/Q100P CD28 Domain 1: CD80 175 CD28 3110 (2.53) 4848 (0.44)3393 (1.93) I67T/L70Q/A91G/T120S Domain 2: ICOSL 201 ICOS/N52H/N57Y/Q100P CD28 Domain 1: ICOSL WT 196 3000 (1.00) 14366 (1.00)4113 (1.00) Domain 2: CD80 WT 152 Domain 1: ICOSL 213 ICOSL/ 10426(3.48) 51286 (3.57) 18680 (4.54) N52S/N57Y/H94D/L96F/ CD28L98F/Q100R/G103E/ F120S Domain 2: CD80 192 CD28 R29H/Y31H/T41G/Y87N/E88G/K89E/D90N/ A91G/P109S Domain 1: ICOSL 213 ICOS/ 17751 (5.92) 29790(2.07) 10637 (2.59) N52S/N57Y/H94D/L96F/ CD28 L98F/Q100R/G103E/ F120SDomain 2: CD80 175 CD28 I67T/L70Q/A91G/T120S Domain 1: ICOSL 199 ICOS/2788 (0.93) 25870 (1.80) 6205 (1.51) N52D CD28 Domain 2: CD80 192 CD28R29H/Y31H/T41G/Y87N/ E88G/K89E/D90N/ A91G/P109S Domain 1: ICOSL 199ICOS/ 2522 (0.84) 13569 (0.94) 5447 (1.32) N52D CD28 Domain 2: CD80 175CD28 I67T/L70Q/A91G/T120S Domain 1: ICOSL 201 ICOS/ 9701 (3.23) 9187(0.64) 5690 (1.38) N52H/N57Y/Q100P CD28 Domain 2: CD80 192 CD28R29H/Y31H/T41G/Y87N/ E88G/K89E/D90N/ A91G/P109S

Example 10 Generation and Assessment of Engineered Cells Expressing aTransmembrane Immunomodulatory Protein

Engineered T cells were generated in which a transmembraneimmunomodulatory protein (TIP) containing an extracellular domain (ECD)containing either a variant CD80 as described above or an ICOSLaffinity-modified IgSF domain was co-expressed with a chimeric antigenreceptor (CAR). The TIP also contained a transmembrane domain and acytoplasmic domain of the corresponding wild-type CD80 or ICOSLtransmembrane protein sequence. The immunomodulatory activity of theengineered cells was compared to cells that only expressed the CAR orcells that co-expressed the corresponding wild-type CD80 or ICOSLtransmembrane protein with the CAR.

The exemplary CD80-TIP was a variant CD80 having an affinity-modifiedIgSF domain containing amino acid mutations in the IgV and IgC domainscorresponding to I67T/L70Q/A91G/T120S with reference to positions in theCD80 extracellular domain set forth in SEQ ID NO:28 and a transmembraneand cytoplasmic domain corresponding to residues 243-288 of SEQ ID NO:1.The amino acid sequence of the exemplary CD80-TIP is set forth in SEQ IDNO:241 and is encoded by the sequence of nucleotides set forth in SEQ IDNO:242. The corresponding wild-type CD80 transmembrane protein had thesequence of amino acids set forth as amino acid residues 35-288 of SEQID NO:1 and encoded by the sequence of amino acids set forth in SEQ IDNO: 251.

The exemplary ICOSL-TIP was a variant ICOSL having an affinity-modifiedIgSF domain containing amino acid mutations in the IgV domaincorresponding to N52H/I143T with reference to positions in the ICOSLextracellular domain set forth in SEQ ID NO:32 and a transmembrane andcytoplasmic domain corresponding to residues 257-302 of SEQ ID NO:5. Theamino acid sequence of the exemplary ICOSL-TIP is set forth in SEQ IDNO:243 and is encoded by the sequence of nucleotides set forth in SEQ IDNO:244. The corresponding wild-type ICOSL transmembrane protein had thesequence of amino acids set forth as amino acid residues 19-302 of SEQID NO:5 and encoded by the sequence of amino acids set forth in SEQ IDNO: 252.

The TIP containing the affinity-modified domain or the wild-typetransmembrane protein containing a corresponding non-affinity modifiedIgSF domain were co-expressed in T cells with a 1^(st) generationchimeric antigen receptor (CAR) containing a CD3zeta intracellularsignaling domain. The 1^(st) generation CAR included an scFv specificfor CD19 (SEQ ID NO:245), a hinge and transmembrane domain derived fromCD8 (SEQ ID NO:246) and an intracellular signaling domain derived fromCD3zeta (set forth in SEQ ID NO:247). The nucleotide sequence encodingthe CD19 scFv-CD3zeta CAR is set forth in SEQ ID NO:248 and the aminoacid sequence of the CD19 scFv-CD3zeta CAR is set forth in SEQ IDNO:479.

Nucleic acid molecules encoding the CAR alone or also encoding one ofthe exemplary TIPs or wild-type transmembrane proteins separated fromthe CAR by a self-cleaving T2A sequence (SEQ ID NO:250 and encoded bythe sequence of nucleotides set forth in SEQ ID NO:249) were generated.Exemplary constructs contained nucleic acid sequences set forth in Table15. As a control, a nucleic acid construct encoding a 2^(nd) generationCAR additionally containing a CD28 costimulatory domain also wasgenerated (CD19 scFv-CD28-CD3zeta).

TABLE 15 Nucleic Acid Constructs CAR T2A Linker TIP (SEQ ID NO) (SEQ IDNO) (SEQ ID NO) CD19 scFv − CD3zeta + − − (248) CD19 scFv − CD3zeta− + + Wildtype CD80 T2A − B7-1 (248) (249) (251) CD19 scFv − CD3zeta + +CD80 TIP T2A − B7-1_TIP (248) (249) (242) CD19 scFv − CD3zeta − + +Wildtype T2A − ICOSL (248) (249) ICOSL (252) CD19 scFv − CD3zeta + +ICOSL TIP T2A − ICOSL_TIP (248) (249) (244)

The nucleic acid molecules were individually cloned into a lentiviralvector, which was used to transduce T cells isolated from human PBMCsamples obtained from three different healthy donors. Lentivirusparticles containing the nucleic acid sequences were produced afterco-transfection of HEK293 cells with the vectors and lentiviruspackaging constructs. The lentivirus particles were collected from theculture medium by ultracentrifugation and titered by qRT-PCR. Humanperipheral blood mononuclear cells (PBMC) were isolated from threenormal blood donors using density sedimentation. The PBMC were culturedovernight with anti-CD3 and anti-CD28 antibodies and IL-2, and thentransduced with the lentivirus preparations at a multiplicity ofinfection of 5:1. The lentiviral vectors encoding the control 2^(nd)generation CAR was only used to transduce cells from one donor.

After two weeks (14 days) of culture, the cells were analyzed forcytotoxicity following co-culture with target antigen-expressing cellsusing the Acea Real-Time Cell Analyzer (RTCA), which measures theimpedance variations in the culture media of a 96-well microelectronicplate (E-plate), and shows the changes in cell number and morphology ina real-time plot. CD19-expressing HeLa target cells (HeLa-CD19) wereseeded into a 96-well E-plate and the impedance of each monolayer wasmonitored for 24 hours using the RTCA system. The engineered T cellswere added to the wells at an effector:target ratio of 10:1 and thewells were monitored for another 48 hours. The results were displayedand recorded as Cell Index (CI) value derived from the change inmeasured electrical impedance and were then ratio transformed bydividing the CI readouts of all wells at all time points over the CIvalue of individual wells at a same time (base-time) to obtain anormalized cell index value representing the percentage of the value atthe base-time (see Zhang et al. “Introduction to the Data Analysis ofthe Roche xCELLigence® System with RTCA Package.” Bioconductor. May 3,2016,bioconductor.org/packages/devel/bioc/vignettes/RTCA/inst/doc/aboutRTCA.pdf.Accessed Sep. 9, 2016). In this assay, a decrease in the impedance of amonolayer reflects killing of the target cells by the transduced cells.

The results showed that decreased impedance was observed in cellsexpressing the 1^(st) generation CAR compared to non-transduced T cells,although the degree of decreased impedance for cells expressing the1^(st) generation CAR was less than cells expressing the 2^(nd)generation CAR. The decreased impedance in cells expressing the 1^(st)generation CAR continued generally for up to the first 8 hours of theassay, while only the 2^(nd) generation CAR-expressing cells continuedto decrease the impedance thereafter.

As shown in FIG. 8, in one donor, each of the cells co-expressing theTIP or corresponding wild-type transmembrane protein with the 1^(st)generation CAR exhibited a greater decrease in impedance, indicatinggreater cytotoxic activity, compared to cells only expressing the 1^(st)generation CAR. Further, the results showed that the cytotoxic activitywas greater in CAR-expressing cells that co-expressed the CD80-TIP orICOSL-TIP relative to CAR-expressing cells that co-expressed thecorresponding wild-type CD80 or ICOSL transmembrane proteins containinga non-affinity modified IgSF domain. The observed results of theseTIP-engineered cells showed that cytotoxic activity in cellsco-expressing the CD80-TIP or ICOSL-TIP with the CAR exhibit increasedactivity to modulate the cytotoxic immune response of antigen-specific Tcells, such as the CAR-expressing T cells.

In the other two donors, the cells expressing the CD80-TIP did notresult in a greater decreased impedance compared to cells expressing thecorresponding wild-type CD80 transmembrane protein. In one donor, therewere not enough cells to transduce with the wild-type transmembraneprotein construct, although in this donor the ICOS-L TIP gave the bestcytotoxicity compared to the other constructs tested. In the otherdonor, the cells expressing the ICOS-L-TIP did not result in a greaterdecreased impedance compared to cells expressing the correspondingwild-type ICOS-L transmembrane protein. In the tested cells, all cellsco-expressing either a CD80-TIP, ICOSL-TIP or corresponding wild typetransmembrane protein with the CAR exhibited greater cytotoxic activitythan cells only expressing the 1st generation CAR. The differences inthe results observed among donors may be related to the differences inthe T cells among the donors, differences in expression levels of thevarious engineered proteins on the surface of the cells, the particularconditions used in this exemplary assay for assessing killing in cells(e.g. assessing Day 14 transduced cells, assessing a singleeffector:target cell ratio) or other factors.

Example 11 Assessment of Binding and Activity of CD80 IgSF DomainVariants

Additional ECD CD80 variants were identified and were used to produceECD-Fc fusion proteins as described in Example 5. Binding studies wereperformed to assess specificity and affinity of CD80 domain variantimmunomodulatory proteins for cognate binding partners substantially asdescribed in Example 6. Exemplary results for the additional CD80 IgSFdomain variants for binding to cell-expressed counter structures andbioactivity from an anti-CD3 coimmobilization assay are set forth inTable 16.

The exemplary amino acid substitutions depicted in Table 16 aredesignated by amino acid position number corresponding to the respectivereference unmodified CD80 ECD sequence set forth in SEQ ID NO:28. Theamino acid position is indicated in the middle, with the correspondingunmodified (e.g. wild-type) amino acid listed before the number and theidentified variant amino acid substitution listed after the number.Column 2 sets forth the SEQ ID NO identifier for the variant ECD foreach variant ECD-Fc fusion molecule. Also shown is the binding activityas measured by the Mean Fluorescence Intensity (MFI) value for bindingof each variant Fc-fusion molecule to cells engineered to express thecognate counter structure ligand and the ratio of the MFI compared tothe binding of the corresponding unmodified ECD-Fc fusion molecule notcontaining the amino acid substitution(s) to the same cell-expressedcounter structure ligand. The functional activity of the variantFc-fusion molecules to modulate the activity of T cells also is shownbased on the calculated levels of IFN-gamma, and the ratio of IFN-gammacompared to the corresponding unmodified (parental) ECD-Fc, in culturesupernatants (pg/ml) generated with the indicated variant ECD-Fc fusionmolecule coimmoblized with anti-CD3. The results show altered, includingincreased, binding affinity of affinity-modified CD80 IgSF domainvariants for at least one cognate counter structure ligand and/orimproved immunological activity.

TABLE 16 CD80 variants selected against CTLA-4 or PD-L1. Moleculesequences, binding data, and costimulatory bioactivity data. Anti-CD3IFN- gamma Coimmobi- CD28 CTLA-4 PD-L1 lization SEQ tfxn tfxn tfxn AssayID MFI MFI MFI pg/ml NO (parental (parental (parental (parental CD80mutation(s) (ECD) ratio) ratio) ratio) ratio) R29D, Y31L, Q33H, K36G,M38I, 446 3536 (0.08) 5731 (0.01) 173405 (0.08) 109 (0.24) T41A, M43R,M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, I118V, T120S, I127T,T130A, K169E R29D, Y31L, Q33H, K36G, M38I, 447 4962 (0.11) 2027 (0.01)626341 (0.11) 162 (0.36) T41A, M43R, M47T, L70Q, E81V, L85R, K89N, A91T,F92P, K93V, R94L, I118V, T120S, I127T, T130A, H18L, R29D, Y31L, Q33H,K36G, 448 3489 (0.08) 2521 (0.01) 215826 (0.08) 206 (0.46) M38I, T41A,M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, I118V, T120S,I127T, T130A, K169E R29D, Y31L, Q33H, K36G, M38I, 449 2736 (0.06) 2493(0.01) 157897 (0.06) 141 (0.31) T41A, M43R, M47T, E81V, L85R, K89N,A91T, F92P, K93V, R94L, I118V, T120S, T130A, K169E, M174T R29D, Y31L,Q33H, K36G, M38I, 450 2393 (0.05) 2663 (0.01) 137062 (0.05) 230 (0.51)T41A, M43R, M47T, N48D, F59L, E81V, L85R, K89N, A91T, F92P, K93V, R94L,I118V, T120S, I127T, T130A, H188D H18R, R29D, Y31L, Q33H, K36G, 451 3023(0.07) 2303 (0.01) 158977 (0.07) 305 (0.68) K37E, M38I, T41A, M43R,M47T, L70Q, E81V, L85R, K89N, A91T, F92P, K93V, R94L, I118V, T120S,T130A, K169E, H188D R29D, Y31L, Q33H, K36G, M38I, 452 2135 (0.05) 2816(0.01) 374117 (0.05) 291 (0.65) T41A, M43R, M47T, L70Q, E81V, L85R,K89N, A91T, F92P, K93V, R94L, I118V, T120S, I127T, T130A, E143G, K169E,M174V, H188D R29D, Y31L, Q33H, K36G, M38I, 446 2157 (0.05) 2819 (0.01)114963 (0.05) 197 (0.44) T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P,K93V, R94L, I118V, T120S, I127T, T130A, K169E R29D, Y31L, Q33H, K36G,M38I, 447 2126 (0.05) 2377 (0.01) 530029 (0.05) 135 (0.30) T41A, M43R,M47T, L70Q, E81V, L85R, K89N, A91T, F92P, K93V, R94L, I118V, T120S,I127T, T130A R29D, 130V, Y31L, Q33H, K36G, 453 1914 (0.04) 2024 (0.01)179536 (0.04) 127 (0.28) M38I, T41A, M43R, M47T, E81V, L85R, K89N, A91T,F92P, K93V, R94L, I118V, T120S, I127T, T130A, H188D R29D, Y31L, Q33H,K36G, M38I, 455 2377 (0.05) 2177 (0.01) 438352 (0.05) 203 (0.45) T41A,M43R, M47T, L70Q, E81V, L85R, K89N, A91T, F92P, K93V, R94L, I118V,T120S, I127T, T130A, K169E R29D, Y31L, Q33H, K36G, M38I, 456 2106 (0.05)2122 (0.01) 14201 (0.05) 226 (0.50) T41A, M43R, M47T, L70Q, E81V, K89N,A91T, F92P, K93V, R94L, I118V, T120S, I127T, T130A R29D, Y31L, Q33H,K36G, M38I, 457 1887 (0.04) 2201 (0.01) 110092 (0.04) 231 (0.51) T41A,M43R, M47T, L85R, K89N, A91T, F92P, K93V, R94L, I118V, T120S, I127T,T130A, K169E, H188D R29D, 130V, Y31L, Q33H, K36G, 453 2060 (0.05) 2385(0.01) 94786 (0.05) 237 (0.53) M38I, T41A, M43R, M47T, E81V, L85R, K89N,A91T, F92P, K93V, R94L, I118V, T120S, I127T, T130A, H188D R29D, Y31L,Q33H, K36G, M38I, 458 2009 (0.04) 2623 (0.01) 110589 (0.04) 165 (0.37)T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, F108L,I118V, T120S, T130A, K169E, H188D R29D, Y31L, Q33H, K36G, M38I, 293 1925(0.04) 2979 (0.01) 379558 (0.04) 213 (0.47) T41A, M43R, M47T, L70Q,E81V, L85R, K89N, A91T, F92P, K93V, R94L, T130A, H188D R29D, Y31L, Q33H,K36G, M38I, 459 2245 (0.05) 2842 (0.01) 631549 (0.05) 118 (0.26) T41A,M43R, M47T, L70Q, E81V, L85R, K89N, A91T, F92P, K93V, R94L, I118V,T120S, T130A, N149D, K169E, H188D H18L, R29D, Y31L, Q33H, K36G, 460 2759(0.06) 2247 (0.01) 760438 (0.06) 157 (0.35) M38I, T41A, M43R, M47T,L70Q, E81V, L85R, K89N, A91T, F92P, K93V, R94L, I118V, T120S, T130A,K169E, H188D R29D, Y31L, Q33H, K36G, M38I, 461 1585 (0.03) 2736 (0.01)456003 (0.03) 278 (0.62) T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P,K93V, R94L, I118V, T120S, I127T, C128Y, T130A, H188D R29D, Y31L, Q33H,K36G, M38I, 297 2633 (0.06) 3379 (0.01) 133095 (0.06) 190 (0.42) T41A,M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94F, T130A, K169E H18L,R29D, Y31L, Q33H, K36G, 462 1732 (0.04) 2082 (0.01) 117465 (0.04) 174(0.39) M38I, T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L,E99D, T130A H18L, R29D, Y31L, Q33H, K36G, 463 2011 (0.04) 2502 (0.01)711479 (0.04) 232 (0.51) M38I, T41A, M43R, M47T, L70Q, E81V, L85R, K89N,A91T, F92P, K93V, R94L, I118V, T120S, T130A, K169E R29D, Y31L, Q33H,K36G, M38I, 300 2026 (0.04) 2443 (0.01) 572017 (0.04) 202 (0.45) T41A,M43R, M47T, L70Q, E81V, L85R, K89N, A91T, F92P, K93I, R94L, L97R, T130AR29D, Y31L, Q33H, K36G, M38I, T41A, M43R, M47T, L70Q, E81V, 301 1296(0.03) 2119 (0.01) 777509 (0.03) 101 (0.22) L85R, K89N, A91T, F92P,K93I, R94L, L97R, T130A, L148S H18L, R29D, Y31L, Q33H, K36G, 302 1188(0.03) 2161 (0.01) 190176 (0.03) 97 (0.22) M38I, T41A, M43R, M47T, E81V,L85R, K89N, A91T, F92P, K93V, R94L, I118V, T120S, I127T, T130A, K169ER29D, Y31L, Q33H, K36G, M38I, 464 1203 (0.03) 1863 (0.01) 217243 (0.03)288 (0.64) T41A, M43R, M47T, I61N, E81V, L85R, K89N, A91T, F92P, K93V,R94F, V104A, I118V, T120S, I126V, T130A R29D, Y31L, Q33H, K36G, M38I,465 1289 (0.03) 2625 (0.01) 124188 (0.03) 111 (0.25) T41A, M43R, M47T,E81V, L85R, K89N, A91T, F92P, K93V, R94F, I118V, T120S, T130A R29D,Y31L, Q33H, K36G, M38I, 466 1228 (0.03) 1973 (0.01) 145285 (0.03) 114(0.25) T41A, M43R, M47T, T62S, E81V, L85R, K89N, A91T, F92P, K93V, R94L,I118V, T120S, T130A, K169E, T175A H18L, R29D, Y31L, Q33H, K36G, 306 1244(0.03) 2091 (0.01) 109646 (0.03) 114 (0.25) M38I, T41A, M43R, M47T,E81V, L85R, K89N, A91T, F92P, K93V, R94L, F116S, T130A, H188D H18L,R29D, Y31L, Q33H, K36G, 467 1221 (0.03) 2251 (0.01) 89654 (0.03) 143(0.32) M38I, T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L,I118V, T120S, I127T, T130A, L142S, H188D C16S, H18L, R29D, Y31L, Q33H,468 1212 (0.03) 1800 (0.01) 4497 (0.03) 247 (0.55) K36G, M38I, T41A,M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, T110A, I118V,H188D R29D, Y31L, Q33H, K36G, M38I, 469 2620 (0.06) 2241 (0.01) 66183(0.06) 125 (0.28) T41A, M43R, M47T, A91G, I118V, T120S, I127T, T130A,H188D R29D, Y31L, Q33H, K36G, M38I, 470 1907 (0.04) 1726 (0.01) 3508(0.04) 224 (0.50) T41A, M43R, M47T, L70Q, D76G, A91G, S103L, I118V,T120S, I127T, T130A Y53C, L85R, K89N, A91T, F92P, 471 1396 (0.03) 1459(0.01) 2552 (0.03) 75 (0.17) K93V, R94L, I118V, T120S, I127T, T130A,K169E T62S, E81V, L85R, K89N, A91T, F92P, 472 2947 (0.06) 2377 (0.01)179622 (0.06) 107 (0.24) K93V, R94L, I118V, T120S, T130A, K169E L70Q,A91G, I118V, T120S, T130A, 417 12262 (0.27) 90597 (0.01) 4168 (0.27) 115(0.25) K169E R29D, Y31L, Q33H, K36G, M38I, 313 1696 (0.04) 9960 (0.01)4067 (0.04) 87 (0.19) T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P,K93V, R94L, S129L, H188D Y53C/L70Q/D90G/T130A/ 473 1990 (0.04) 3106(0.01) 3289 (0.04) 122 (0.27) N149D/N152T/H188D WT CD80 28 45607 (1.00)883950 (1.00) 93079 (1.00) 451 (1.00) H18L, R29D, Y31L, Q33H, K36G, 47416232 (0.26) 140241 (0.88) 182403 (2.61) 108 (0.96) M38I, T41A, M43R,M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, I118V, T120S, I127T,T130A, H188D K89E, T130A 73 46923 (0.75) 225651 (1.41) 196544 (2.82) 317(2.81) K89E, K93E, T130A 316 39137 (0.63) 181037 (1.13) 206713 (2.96)571 (5.06) K89E, T130A 73 61349 (0.99) 156244 (0.98) 126961 (1.82) 2539(22.53) WT CD80 28 62220 (1.00) 160148 (1.00) 69786 (1.00) 113 (1.00)S21P, R29D, Y31L, Q33H, K36G, 317 4467 (0.20) 4434 (0.02) 146638 (5.87)137 (0.65) M38I, T41A, M43R, M47T, N48I, V68A, E81V, L85R, K89N, A91T,F92P, K93V, R94L,P109H, I126L, K169I H18L, R29D, Y31L, Q33H, K36G, 3184523 (0.21) 4565 (0.02) 4731 (0.19) 18 (0.09) M38I, T41A, M43R, M47T,P74L, Y80N, E81V, L85R, K89N, A91T, F92P, K93V, R94L, L97R R29D, Y31L,Q33H, K36G, M38I, 319 4675 (0.21) 4686 (0.02) 4098 (0.16) 54 (0.26)T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, S21P, P74L,Y80N, D90N, T130A, N149S, E162G R29D, Y31L, Q33H, K36G, M38I, 320 4413(0.20) 4618 (0.02) 221788 (8.88) 51 (0.24) T41A, M43R, M47T, E81V, L85R,K89N, A91T, F92P, K93V, R94L, H18L, V68M, T130A R29D, Y31L, Q33H, K36G,M38I, 321 4354 (0.20) 4413 (0.02) 201513 (8.06) 80 (0.38) T41A, M43R,M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, V68M, T130A, N149S,R190S R29D, Y31L, Q33H, K36G, M38I, 322 4381 (0.20) 4491 (0.02) 5075(0.20) 8 (0.04) T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V,R94L, H18L, P74L, Y80N, T130A, R190S C16G, V22A, R29D, Y31L, Q33H, 3234459 (0.20) 4582 (0.02) 4383 (0.18) 45 (0.21) K36G, M38I, T41A, M43R,M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, V68M, D76G, I118T,T130A, S140T, N149S, K169I, H178R, N192D R29D, Y31L, Q33H, K36G, M38I,324 4371 (0.20) 4613 (0.02) 247135 (9.89) 61 (0.29) T41A, M43R, M47T,E81V, L85R, K89N, A91T, F92P, K93V, R94F, E117V, I118T, N149S, S168G,H188Q V22A, R29D, Y31L, Q33H, K36G, 325 4222 (0.19) 4381 (0.022) 235307(9.42) 96 (0.46) M38I, T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P,K93V, R94L, V68M, T130A R29D, Y31L, Q33H, K36G, M38I, 326 4216 (0.19)4305 (0.022) 149085 (5.97) 30 (0.14) T41A, M43R, M47T, E81V, L85R, K89N,A91T, F92P, K93V, R94F, N64S, I118T, T130A, N149S, K169I R29D, Y31L,Q33H, K36G, M38I, 327 4361 (0.20) 4459 (0.022) 95558 (3.82) 88 (0.42)T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, V22A, V68M,D115G, I118T, T130A, G133D, N149S S129P 328 4458 (0.20) 5914 (0.03) 6669(0.27) 0 (0.0) A91G, S129P 329 5144 (0.23) 26635 (0.12) 13569 (0.54) 0(0.0) I69T, L70Q, A91G, T120S 330 74296 (3.37) 203363 (0.89) 6242 (0.25)509 (2.42) S129P 328 5086 (0.23) 8957 (0.04) 6603 (0.26) 125 (0.60)Y31H, S129P 331 4553 (0.21) 4874 (0.02) 5788 (0.23) 76 (0.36) WT CD80 2822053 (1.00) 227691 (1.00) 24989 (1.00) 211 (1.00) T28A, R29D, Y31L,Q33H, K36G, 332 2636 (0.17) 2169 (0.03) 99658 (9.45) 116 (0.62) M38I,T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, V104L,T130A, N149S H18L, R29D, Y31L, Q33H, K36G, 333 2680 (0.17) 2165 (0.03)107747 (10.22) 125 (0.68) M38I, T41A, M43R, M47T, E81V, L85R, K89N,A91T, F92P, K93V, R94L, L97R, N149S, H188Q H18L, R29D, Y31L, Q33H, K36G,475 2482 (0.16) 2179 (0.03) 100116 (9.50) 96 (0.52) M38I, T41A, M43R,M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, L97R, T130A, N149S R29D,Y31L, Q33H, K36G, M38I, 335 2455 (0.15) 2084 (0.03) 11855 (1.12) 132(0.71) T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, H18L,V68A, T130A, N149S, T154I R29D, Y31L, Q33H, K36G, M38I, 336 2804 (0.18)2310 (0.03) 216297 (20.51) 191 (1.03) T41A, M43R, M47T, E81V, L85R,K89N, A91T, F92P, K93V, R94L, A12G, V68A, L97R, T130A, L183H R29D, Y31L,Q33H, K36G, M38I, 337 2460 (0.15) 2188 (0.03) 109263 (10.36) 150 (0.81)T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, I118T,T130A, S140T, N149S, K169S R29D, Y31L, Q33H, K36G, M38I, 338 2569 (0.16)2198 (0.03) 100074 (9.49) 130 (0.70) T41A, M43R, M47T, E81V, L85R, K89N,A91T, F92P, K93V, R94L, I118T, T130A, N149S, K169I, Q193L R29D, Y31L,Q33H, K36G, M38I, 339 2500 (0.16) 2188 (0.03) 147900 (14.03) 124 (0.67)T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94L, V22A, I118T,T130A, N149S R29D, Y31L, Q33H, K36G, M38I, 340 2615 (0.16) 2210 (0.03)118150 (11.21) 89 (0.48) T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P,K93V, R94L, I118T, T130A, N149S R29D, Y31L, Q33H, K36G, M38I, 341 2444(0.15) 2246 (0.03) 115420 (10.95) 101 (0.55) T41A, M43R, M47T, E81V,L85R, K89N, A91T, F92P, K93V, R94L, I118T, T130A, N149S, K169I R29D,Y31L, Q33H, K36G, M38I, 342 2378 (0.15) 2123 (0.03) 112712 (10.69) 114(0.61) T41A, M43R, M47T, E81V, L85R, K89N, A91T, F92P, K93V, R94F,T130A, N149S, K169I I118T, C128R 343 3093 (0.19) 3180 (0.03) 2620 (0.25)122 (0.66) Q27R, R29C, M42T, S129P, E160G 344 2827 (0.18) 2623 (0.03)2326 (0.22) 139 (0.75) S129P, T154A 345 3062 (0.19) 2622 (0.03) 2606(0.25) 156 (0.84) WT CD80 28 15948 (1.00) 75099 (1.00) 10544 (1.00) 185(1.00)

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

What is claimed is:
 1. A method of treating cancer comprisingadministering to a subject with cancer an effective amount of ahomodimer of a CD80-Fc immunomodulatory protein of the formulaCD80-linker-Fc, wherein: CD80 is a variant CD80 polypeptide comprising asequence of amino acids that exhibits at least 90% sequence identity toan unmodified CD80 polypeptide set forth in SEQ ID NO: 28, or a portionof SEQ ID NO:28 comprising an IgV domain, wherein the IgV domain isamino acids 35-135, 35-138, 37-138 or 35-141 of SEQ ID NO:1; the variantCD80 polypeptide comprises the amino acid substitution V68M; and whereinthe variant CD80 polypeptide specifically binds to the ectodomain ofhuman PD-L1 with increased binding affinity compared to the binding ofthe unmodified CD80 polypeptide to the ectodomain of human PD-L1.
 2. Themethod of claim 1, wherein the variant CD80 polypeptide comprises up to10 amino acid substitutions.
 3. The method of claim 1, wherein thevariant CD80 polypeptide binds non-competitively to the ectodomain ofCD28 and PD-L1.
 4. The method of claim 1, wherein the Fc domain is an Fcdomain from IgG1 or is a variant IgG1 Fc domain with reduced effectorfunction.
 5. The method of claim 4, wherein the variant IgG1 Fc domaincomprises the amino acid substitutions R292C/N297G/V302C.
 6. The methodof claim 4, wherein the variant IgG1 Fc domain comprises the amino acidsubstitutions L234A/L235E/G237A.
 7. The method of claim 1, wherein theFc domain is an Fc domain of IgG2.
 8. The method of claim 1, wherein theFc domain is an Fc domain of IgG4 or is a variant IgG4 Fc domaincontaining the S228P mutation.
 9. The method of claim 1, wherein thevariant CD80 polypeptide comprises a sequence of amino acids thatexhibits at least 90% sequence identity to amino acids 35-141 of SEQ IDNO:1.
 10. The method of claim 1, wherein the variant CD80 polypeptidecomprises a sequence of amino acids that exhibits at least 95% sequenceidentity to amino acids 35-141 of SEQ ID NO:1.
 11. A method of treatingcancer comprising administering to a subject with cancer an effectiveamount of a homodimer of a CD80-Fc immunomodulatory protein of theformula CD80-linker-Fc, wherein: CD80 is a variant CD80 polypeptidecomprising a sequence of amino acids that exhibits at least 90% sequenceidentity to amino acids 35-141 of SEQ ID NO:1 and comprises the aminoacid substitution V68M; and the Fc domain is a variant IgG1 Fc domainwith reduced effector function.
 12. The method of claim 11, wherein thevariant Fc domain comprises the amino acid substitutionsR292C/N297G/V302C.
 13. The method of claim 11, wherein the variant Fcdomain comprises the amino acid substitutions L234A/L235E/G237A.
 14. Themethod of claim 11, wherein the variant CD80 polypeptide comprises asequence of amino acids that exhibits at least 95% sequence identity toamino acids 35-141 of SEQ ID NO:1.
 15. The method of claim 11, whereinthe variant CD80 polypeptide binds non-competitively to the ectodomainof CD28 and PD-L1.
 16. The method of claim 11, wherein the variant CD80polypeptide specifically binds to the ectodomain of human PD-L1 withincreased binding affinity compared to the binding of the unmodifiedCD80 polypeptide to the ectodomain of human PD-L1.
 17. A method oftreating cancer comprising administering to a subject with cancer aneffective amount of a homodimer of a CD80-Fc immunomodulatory protein ofthe formula CD80-linker-Fc, wherein: CD80 is a variant CD80 polypeptidecomprising a sequence of amino acids that exhibits at least 95% sequenceidentity to amino acids 35-141 of SEQ ID NO:1 and comprises the aminoacid substitution V68M; and the Fc domain is a variant IgG1 Fc domaincomprising the amino acid substitutions L234A/L235E/G237A.
 18. Themethod of claim 17, wherein the variant CD80 polypeptide bindsnon-competitively to the ectodomain of CD28 and PD-L1.
 19. The method ofclaim 17, wherein the variant CD80 polypeptide specifically binds to theectodomain of human PD-L1 with increased binding affinity compared tothe binding of the unmodified CD80 polypeptide to the ectodomain humanPD-L1.